Method for selecting and producing vaccine components and vaccines based thereon

ABSTRACT

The invention relates to the field of biology, more specifically to the field of immunology and microbiology. The invention further relates to the field of vaccines against microbial infections and especially bacterial vaccines, in particular to pneumococcal vaccines. More in particular, the invention relates to means and methods to identify, select and isolate a vaccine component for passive and/or active immunisation against a microorganism that can be killed by opsonophagocytic cells. The invention relates to a method to identify an opsonophagocytosis inducing antigen as a vaccine component for immunisation against a microorganism. The invention describes three pneumococcal proteins SlrA, IgAl proteinase, and PsaA, and their use as a vaccine component with or without PpmA. The invention also discloses the use of antibodies against said proteins for passive immunization and diagnosis.

This application is the U.S. National Phase of International Application Number PCT/NL2004/000338 filed on 17 May 2004, which is incorporated herein by reference.

INCORPORATION OF SEQUENCE LISTING

Incorporated herein by reference in its entirety is the Sequence Listing for the above-identified Application. The Sequence Listing is disclosed in a computer-readable ASCII text file titled “Sequence Listing.txt,” created on. The sequence.txt file is bytes in size.

The invention relates to the field of biology, more specifically to the field of immunology and microbiology. The invention further relates to the field of vaccines against microbial infections and more in particular bacterial vaccines such as pneumococcal vaccines. More in particular, the invention relates to means and methods to identify, select and isolate a vaccine component to produce a vaccine for passive and/or active immunization against a microorganism that can be recognized and preferably taken up and more preferably killed by opsonophagocytic cells.

Streptococcus pneumoniae is the leading etiological agent of severe infections such as pneumonia, septicemia, meningitis and otitis media, and causes over three million deaths per year worldwide, of which a million are children. The problem of antimicrobial resistance in Streptococcus pneumoniae has been enhanced by the successful spread of a limited number of internationally recognized multi-resistant strains. These strains have complicated the treatment of pneumococcal disease, and this has further emphasized the need for vaccination as an alternative means of preventing the large-scale morbidity and mortality associated with pneumococcal infection. The initial attempt to develop an anti-pneumococcal vaccine that consisted of 23 out of 90 of the most prevalent capsular polysaccharides (Pneumovax 23©) was unsuccessful, since the vaccine polysaccharides were unable to elicit an immune response in people at most risk of pneumococcal infection: young children, immuno-compromised patients, and the elderly. Technical advances have allowed the linkage of capsular polysaccharides to highly immunogenic carrier proteins. The implementation of this technology using a vaccine consisting of tetanus toxoid linked to Haemophilus influenzae type B-polysaccharide has led to undetectable levels of carriage of Haemophilus influenzae type B, and an elimination of disease caused by this organism in vaccinated populations. Recently, a polysaccharide conjugate pneumococcal vaccine (Prevnar©) has been licensed in the USA by Wyeth Lederle Vaccines. Prevnar© has been released in The Netherlands in 2001. The vaccine formulation is immunogenic in children, and consists of 7 of the most prevalent capsule polysaccharides conjugated to a highly immunogenic (non-pneumococcal) carrier protein. However, unlike the Haemophilus influenzae vaccine, this vaccine covers only 7 out of 90 of the known pneumococcal capsule serotypes, and clinical data from phase three trials conducted in the USA, Israel, and South Africa have shown that the use of this vaccine causes a shift in colonization by non-vaccine serotypes in place of vaccine serotypes (1, 2, 3, 4, 5). As a result of this apparent weakness associated with narrow spectrum polysaccharide based antigens, attention has been focused on the non-polysaccharide capsule related pneumococcal proteins, many of which are involved in the pathogenesis of infections. These proteins are considered to be interesting components for future vaccines, since they cover all serotypes of pneumococci. The protein loci are less variable than those encoding the genes for capsular polysaccharide production, and they are more likely to be able to elicit an immune response in children (6, 7, 8, 9).

Further all the vaccines that have been developed thus far are systemic vaccines, i.e. vaccines that have to be injected. A mucosal vaccine that could e.g. be administered intranasally would be preferred for ease of administration, especially in children.

In our ongoing studies on the molecular epidemiology and pathogenesis of Streptococcus pneumoniae, we have developed a method for the extraction of surface associated pneumococcal proteins and the subsequent testing of said proteins for binding to antibodies. The antibody-antigen complex is identified, said antigen is isolated and characterized and antibodies, produced against said antigen, are subsequently tested for their opsonizing ability for the microorganism. The antigen, which is binding to an antibody with opsonizing ability for the microorganism having such an antigen on its surface, is identified by for example high-resolution two dimensional PAGE (polyacrylamide gel electrophorsesis) and mass spectrometric identification of the gel spots. This method has provided a means by which we isolated and identified a pneumococcal maturation protein (PpmA) with vaccine potential (10, 11).

The techniques used in the identification of surface-associated pneumococcal proteins, the sequence of ppmA and its protein product (PpmA) and its therapeutic potential are filed under European Patent application nr. 99202640.1.

Many microorganisms are characterized in that they are killed by a phagocytic cell after phagocytosis. Many microorganisms need to be opsonized by opsonins to enhance their phagocytosis. Opsonization is the process of making a microorganism more susceptible for the uptake by a phagocyte. In said process, opsonizing antibodies and/or proteins bind to said microorganism, thereby facilitating the uptake of said microorganism by said phagocyte. Opsonophagocytosis is measured in living phagocytic cells and comprises measuring the uptake by phagocytic cells of microorganisms pretreated with opsonizing antibodies. With this method a selection can be made for surface antigens i.e. antigens naturally or artificially associated with the surface of a microorganism that specifically induce opsonophagocytosis. Of course, surface antigens with opsonophagocytosis inducing ability can be selected from a collection of surface proteins. This typically implies that said surface proteins are isolated from microorganisms and subsequently are purified and tested.

DESCRIPTION OF THE INVENTION

In the present application we disclose a method for identifying surface proteins with opsonophagocytosis inducing ability that does not require the above described mixture of surface antigens as used in the patent application 99202640.1. We hereby disclose a method to combine selection, especially in silico selection with a functional test for opsonophagocytosis. By searching in silico for sequence motifs coding for a membrane anchor, or more preferably for a cell wall binding domain, a protein with opsonophagocytic potential is identified. Said in silico selection is followed by recombinant production of at least part of said selected protein. Said recombinant protein is subsequently used to immunize animals, and/or to contact a collection of antibodies. In mice, active and passive immunization procedures with said recombinant protein or with antibodies against said protein are performed to test the opsonizing potential of said recombinant protein. Protective ability of the selected protein or part thereof may be measured in a vaccination challenge experiment.

With this method, three proteins have been identified as possible phagocytosis inducing vaccine components and are disclosed herein. One of said proteins is a protein called pneumococcal IgA1 protease (IgA1 proteinase), a protease that can convert human dimer IgA molecules into monomers. Another protein is a peptidyl prolyl isomerase, which we named streptococcal lipoprotein rotamase A (SlrA). Yet another protein is pneumococcal surface adhesion protein A (PsaA)

PpmA protein has been classified as an isomerase enzyme. In our search for other proteins with a homologous function on the surface of the microorganism, we found a protein with a peptidyl prolyl isomerase activity, SlrA. In this application, homologous is defined as sequential homology and/or functional homology.

SlrA, although belonging to another family of proteins than PpmA also appeared to be an opsonophagocytosis-inducing antigen. Surprisingly, abovementioned proteins have not been identified by any other method as being opsonophagocytosis enhancing proteins. For example, there was no correlation between antibody titers against SlrA, or IgA1 proteinase and the first episode of acute otitis media (middle ear infection) in children in a large serological survey in Finland as is described in example one. In the absence of such a correlation, these proteins were not considered to be important for the defense against streptococcal infection.

Since the opsonophagocytic activity correlates with in vivo protection, these data show that SlrA, IgA1 proteinase and PsaA have potential to elicit immune protection. Loss of virulence of microorganisms is often tested in vitro by investigating the replication of a microorganism missing said protein. The absence of SlrA, IgA1 proteinase, or PsaA in recombinant strains of Streptococcus pneumoniae did not influence the in vitro characteristics of said microorganism. Therefore, said proteins would not have been selected as vaccine components by methods that are state of the art.

We disclose, in one embodiment, a method to identify an opsonophagocytosis inducing antigen as a vaccine component for immunization against a microorganism, comprising selecting (in silico) via a molecule associated with the surface of a microorganism such as a membrane anchoring motif and/or a cell wall binding domain, a nucleotide sequence encoding at least part of a bacterial protein, and producing at least part of said protein, and contacting a collection of antibodies to said protein and testing for binding between at least one antibody and at least one antigen, and measuring the opsonizing ability of said binding antibody for a microorganism having such an antigen.

The motifs which can be used for the in silico selection comprise, for example, a lipobox (anchoring to membrane via a lipid group), a peptidoglycan binding domain(PBD), a chohne binding domain (CBD) or a LPxTG motief (cell wall binding domain). The above mentioned motifs are characterized as follows:

-   PBD: [YV]-X _((0,4))-G-D-[ST]-[VLIA]-X _((0,2))-[VLIA] (SEQ ID NO:1) -   CBD: G-X _((0,5))-G-X-[WYI]-[WYT]-[YVL]-[FV] (SEQ ID NO:2) -   Lipobox (+signal peptide):<M-X     _((1,10))-[RK]-X-{DERK}_((7,17))-[LVTIMG]-[ASTIVGMLCPFL]-[AGLISVTFP]-C     (SEQ ID NO: 3) -   LPxTG: L-P-X-T-G (SEQ ID NO: 4)     in which X denotes any naturally occurring amino acid, and in which     the square brackets indicate one position which can be filled with     any of the amino acids indicated between the square brackets. The     numbers is subscript indicate that the corresponding amino acid is     repeated the indicated number of times.

Of course, said collection of antibodies can be produced by raising antibodies in an animal, by immunizing for example a rabbit or a mouse or a rat or another animal with at least part of said selected protein of said microorganism. Therefore, this application also teaches a method to identify an opsonophagocytosis inducing antigen as a vaccine component for immunisation against a microorganism as described above wherein said collection of antibodies is raised by immunizing an animal with at least one antigen of said microorganism. Antibodies against said proteins may also be selected from pre-existing collections of antibodies or functional parts, derivatives or analogues thereof. This method can be applied to any microorganism for which the defense by the immune system depends on opsonophagocytosis. Therefore, the application teaches a method to identify an opsonophagocytosis inducing antigen as a vaccine component for immunisation against a microorganism as described, wherein said microorganism is for example a member of the genus Streptococcus, for example a member of the species of Streptococcus pneumoniae, or Streptococcus viridans, or Streptococcus canis, or Streptococcus suis, or a member of the genus Staphylococcus, for example a member of the species Staphylococcus aureus, or Staphylococcus epidermidis, or a coagulase negative Staphylococcus, or a member of the species Moraxella catarrhalis, or Neisseria meningitidis, or Neisseria ghonorrhoeae, or Helicobacter pylori, or Haemophilus influenzae or Campylobacter jejuni, or a member of the genus Pseudomonas, or the genus Bacillus.

Once identified, said partially homologous protein or a part thereof is recombinantly produced and tested for its capability to induce opsonophagocytosis. Therefore, this application also teaches a nucleotide sequence at least encoding for a functional part of a protein, said protein inducing opsonophagocytosis of a microorganism having such a protein, wherein said protein is partially homologous with a protein selected by the method as described above. Said nucleotide sequence can be incorporated into a recombinant vector and herewith, recombinant cells can be provided. Said recombinant cell produces protein based on said nucleotide sequence, therefore, this application teaches a recombinant vector comprising a nucleotide sequence at least encoding for a functional part of a protein, said protein inducing opsonophagocytosis of a microorganism having such a protein, wherein said protein is partially homologous with a protein selected by the method as described above. The application also teaches a recombinant cell expressing at least part of a nucleotide, and a purified protein, produced by said recombinant cell. Said antigen or protein or a part thereof can be used as a vaccine component for immunisation against a microorganism, therefore this application teaches a vaccine component for immunisation against a micro organism which can be killed by opsonophagocytic cells, said vaccine component comprising said protein, and/or an antigenic part thereof. In one embodiment, said vaccine component may comprise at least a functional part of streptococcal lipoprotein rotamase A (SlrA) of Streptococcus pneumoniae. Therefore, this patent application discloses said vaccine component, comprising at least a functional part of streptococcal lipoprotein rotamase A (SlrA) of Streptococcus pneumoniae. In another embodiment, said vaccine component may comprise at least a functional part of pneumococcal IgA1 protease (IgA1) of Streptococcus pneumoniae. Therefore, this patent application discloses said vaccine component, comprising at least a functional part of pneumococcal IgA1 protease (IgA1) of Streptococcus pneumoniae.

In yet another embodiment, said vaccine component may comprise at least a functional part of pneumococcal surface adhesion protein A (PsaA) of Streptococcus pneumoniae. Therefore, this patent application discloses said vaccine component, comprising at least a functional part of pneumococcal surface adhesion protein A (PsaA) of Streptococcus pneumoniae.

With the wording “at least a functional part” of a protein it is meant that the minimal amount of amino acid is taken which is still able to raise an immune response in the vaccinate subject. Also the term “antigenic part” is used in this specification to identify such a functional part of a protein. Generally, such parts will at least be more than 10 amino acids, and preferably about 50-100 amino acids.

We disclose in this patent application the protective ability of said proteins by active and passive immunization in a mouse model both with systemic vaccination and mucosal vaccination.

It may be advantageous to combine more than one surface protein or a functional part thereof in a vaccine, and also to combine at least one surface protein or functional part thereof with PpmA or with another protein to induce an even better immunity. Therefore, in another embodiment, said vaccine component may also comprise at least an antigenic part of pneumococcal maturation protein A (PpmA) of Streptococcus pneumoniae or another protein. With said vaccine components, a vaccine may be produced for the treatment of an infection by a microorganism, which can be killed by opsonophagocytic cells.

Therefore, this application teaches a vaccine comprising at least one of said vaccine components and/or a homologous and/or functionally homologous protein or protein fragment thereof for the treatment of an infection by a microorganism that can be killed by opsonophagocytic cells. Of course, one may formulate said vaccine with a pharmaceutically acceptable carrier and/or adjuvant. Preferably said adjuvant comprises cell wall material of Gram positive bacteria, more preferable it comprises co-called ghosts (empty bacterial cells, which still have an intact cell wall structure). It is further preferable to couple the antigens to such ghosts, for example by using protein anchors, such as the AcmA protein anchor. Other adjuvants may be chosen from adjuvant preparations known in the art, such as emulsions with vitamin E. Therefore, this application also teaches said vaccine, also comprising a pharmaceutically acceptable carrier and/or adjuvant.

For passive immunization, specific antibodies against each of said proteins were administered to mice and the protection of said passive immunity against a challenge infection with virulent Streptococcus pneumoniae was measured. Of course, this method can now be applied to all microorganisms for which the immune response by the host is based on killing of said microorganism by phagocytes, for example but not restricted to for example a member of the genus Strepococcus, for example a member of the species of Strepococcus pneumoniae, or Strepococcus viridans, or Strepococcus canis, or Strepococcus suis, or a member of the genus Staphylococcus, for example a member of the species Staphylococcus aureus, or Staphylococcus epidermidis, or a coagulase negative Staphylococcus, or a member of the species Moraxella catarrhalis, or Neisseria meningitidis, or Neisseria ghonorrhoeae, or Helicobacter pylori, or Haemophilus influenzae or Cainpylobacter jejuni, or a member of the genus Pseudomonas, or the genus Bacillus.

As used herein, the terms “antibody” and “antibodies” refer to monoclonal antibodies, multispecific antibodies (e.g., bi-specific), human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, single-chain Fvs (scFv), single chain antibodies, synthetic antibodies, single domain antibodies, Fab fragments, F(ab) fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

Antibodies of the invention include, but are not limited to, monoclonal antibodies, multispecific antibodies, synthetic antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab′) fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. In particular, antibodies of the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to a vaccine component according to the invention. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

The antibodies of the invention may be from any animal origin including birds and mammals (e.g., human, murine, donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken). Preferably, the antibodies of the invention are human or humanized monoclonal antibodies. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries (including, but not limited to, synthetic libraries of immunoglobulin sequences homologous to human immunoglobulin sequences) or from mice that express antibodies from human genes.

In certain embodiments, high potency antibodies can be used in the methods of the invention. For example, high potency antibodies can be produced by genetically engineering appropriate antibody gene sequences and expressing the antibody sequences in a suitable host. The antibodies produced can be screened to identify antibodies with, e.g., high kon values in a BIAcore assay (see below).

In certain embodiments, an antibody to be used with the methods of the present invention or fragment thereof has an affinity constant or Ka (kon/koff) of at least 102 M-1, at least 5×102 M-1, at least 103 M-1, at least 5×103 M-1, at least 104 M-1, at least 5×104 M-1, at least 105 M-1, at least 5×105 M-1, at least 106 M-1, at least 5×106 M-1, at least 107 M-1, at least 5×107 M-1, at least 108 M-1, at least 5×108 M-1, at least 109 M-1, at least 5×109 M-1, at least 1010 M-1, at least 5×1010 M-1, at least 1011 M-1, at least 5×1011 M-1, at least 1012 M-1, at least 5×1012 M-1, at least 1013 M-1, at least 5×1013 M-1, at least 1014 M-1, at least 5×1014 M-1, at least 1015 M-1, or at least 5×1015 M-1. In yet another embodiment, an antibody to be used with the methods of the invention or fragment thereof has a dissociation constant or Kd (koff/kon) of less than 10-2 M, less than 5×10-2 M, less than 10-3 M, less than 5×10-3 M, less than 10-4 M, less than 5×10-4 M, less than 10-5 M, less than 5×10-5 M, less than 10-6 M, less than 5×10-6 M, less than 10-7 M, less than 5×10-7 M, less than 10-8 M, less than 5×10-8 M, less than 10-9 M, less than 5×10-9 M, less than 10-10 M, less than 5×10-10 M, less than 10-11 M, less than 5×10-11 M, less than 10-12 M, less than 5×10-12 M, less than 10-13 M, less than 5×10-13 M, less than 10-14 M, less than 5×10-14 M, less than 10-15 M, or less than 5×10-15 M.

In certain embodiments, an antibody to be used with the methods of the invention or fragment thereof that has a median effective concentration (EC50) of less than 0.01 nM, less than 0.025 nM, less than 0.05 nM, less than 0.1 nM, less than 0.25 nM, less than 0.5 nM, less than 0.75 nM, less than 1 nM, less than 1.25 nM, less than 1.5 nM, less than 1.75 nM, or less than 2 nM, in an in vitro microneutralization assay. The median effective concentration is the concentration of antibody or antibody fragments that neutralizes 50% of the infective micro-organism in an in vitro microneutralization assay. In a preferred embodiment, an antibody to be used with the methods of the invention or fragment thereof has an EC50 of less than 0.01 nM, less than 0.025 nM, less than 0.05 nM, less than 0.1 nM, less than 0.25 nM, less than 0.5 nM, less than 0.75 nM, less than 1 nM, less than 1.25 nM, less than 1.5 nM, less than 1.75 nM, or less than 2 nM, in an in vitro microneutralization assay.

The antibodies to be used with the methods of the invention include derivatives that are modified, i.e, by the covalent attachment of any type of molecule to the antibody. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, synthesis in the presence of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.

The present invention also provides antibodies of the invention or fragments thereof that comprise a framework region known to those of skill in the art. In certain embodiments, one or more framework regions, preferably, all of the framework regions, of an antibody to be used in the methods of the invention or fragment thereof are human. In certain other embodiments of the invention, the fragment region of an antibody of the invention or fragment thereof is humanized. In certain embodiments, the antibody to be used with the methods of the invention is a synthetic antibody, a monoclonal antibody, an intrabody, a chimeric antibody, a human antibody, a humanized chimeric antibody, a humanized antibody, a glycosylated antibody, a multispecific antibody, a human antibody, a single-chain antibody, or a bispecific antibody.

In certain embodiments of the invention, the antibodies to be used with the invention have half-lives in a mammal, preferably a human, of greater than 12 hours, greater than 1 day, greater than 3 days, greater than 6 days, greater than 10 days, greater than 15 days, greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. Antibodies or antigen-binding fragments thereof having increased in vivo half-lives can be generated by techniques known to those of skill in the art. For example, antibodies or antigen-binding fragments thereof with increased in vivo half-lives can be generated by modifying (e.g., substituting, deleting or adding) amino acid residues identified as involved in the interaction between the Fc domain and the FcRn receptor (see, e.g., PCT Publication No. WO 97/34631 and U.S. Patent Application No.: 10/020,354, entitled “Molecules with Extended Half-Lives, Compositions and Uses Thereof”, filed Dec. 12, 2001, by Johnson et al., which are incorporated herein by reference). Such antibodies or antigen-binding fragments thereof can be tested for binding activity to the vaccine components of the invention as well as for in vivo efficacy using methods known to those skilled in the art, for example, by immunoassays described herein.

Further, antibodies or antigen-binding fragments thereof with increased in vivo half-lives can be generated by attaching to said antibodies or antibody fragments polymer molecules such as high molecular weight polyethyleneglycol. (PEG). PEG can be attached to said antibodies or antibody fragments with or without a multifunctional linker either through site-specific conjugation of the PEG to the N- or C-terminus of said antibodies or antibody fragments or via epsilon-amino groups present on lysine residues. Linear or branched polymer derivatization that results in minimal loss of biological activity will be used. The degree of conjugation will be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibodies. Unreacted PEG can be separated from antibody-PEG conjugates by, e.g., size exclusion or ion-exchange chromatography. PEG-derivatizated antibodies or antigen-binding fragments thereof can be tested for binding activity to the vaccine components of the invention as well as for in vivo efficacy using methods known to those skilled in the art, for example, by immunoassays described herein.

In certain embodiments, the antibodies to be used with the methods of the invention are fusion proteins comprising an antibody or fragment thereof that immunospecifically binds to a vaccine component of the invention and a heterologous polypeptide. Preferably, the heterologous polypeptide that the antibody or antibody fragment is fused to is useful for targeting the antibody to cells which are prone to infection.

In certain embodiments, antibodies to be used with the methods of the invention or fragments thereof disrupt or prevent the interaction between an microbial antigen and its host cell receptor.

In certain embodiments, antibodies to be used with the methods of the invention are single-chain antibodies. The design and construction of a single-chain antibody is described in Marasco et al, 1993, Proc Natl Acad Sci 90:7889-7893, which is incorporated herein by reference.

In certain embodiments, the antibodies to be used with the invention bind to an intracellular epitope, i.e., are intrabodies. An intrabody comprises at least a portion of an antibody that is capable of immunospecifically binding an antigen and preferably does not contain sequences coding for its secretion. Such antibodies will bind its antigen intracellularly. In one embodiment, the intrabody comprises a single-chain Fv (“sFv”). sFv are antibody fragments comprising the VH (variable heavy) and VL (variable light) domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994). In a further embodiment, the intrabody preferably does not encode an operable secretory sequence and thus remains within the cell (see generally Marasco, Wash., 1998, “Intrabodies: Basic Research and Clinical Gene Therapy Applications” Springer:New York).

Generation of intrabodies is well-known to the skilled artisan and is described for example in U.S. Pat. Nos. 6,004,940; 6,072,036; 5,965,371, which are incorporated by reference. Further, the construction of intrabodies is discussed in Ohage and Steipe, 1999, J. Mol. Biol. 291:1119-1128; Ohage et al., 1999, J. Mol. Biol. 291:1129-1134; and Wirtz and Steipe, 1999, Protein Science 8:2245-2250. Recombinant molecular biological techniques such as those described for recombinant production of antibodies (see below) may also be used in the generation of intrabodies.

In one embodiment, intrabodies of the invention retain at least about 75% of the binding effectiveness of the complete antibody (i.e., having constant as well as variable regions) to the antigen. More preferably, the intrabody retains at least 85% of the binding effectiveness of the complete antibody. Still more preferably, the intrabody retains at least 90% of the binding effectiveness of the complete antibody. Even more preferably, the intrabody retains at least 95% of the binding effectiveness of the complete antibody.

In producing intrabodies, polynucleotides encoding the variable region for both the VH and VL chains of interest can be cloned by using, for example, hybridoma mRNA or splenic mRNA as a template for PCR amplification of such domains (Huse et al., 1989, Science 246:1276). In one preferred embodiment, the polynucleotides encoding the VH and VL domains are joined by a polynucleotide sequence encoding a linker to make a single chain antibody (sFv). The sFv typically comprises a single peptide with the sequence VH-linker-VL or VL-linker-VH. The linker is chosen to permit the heavy chain and light chain to bind together in their proper conformational orientation (see for example, Huston, et al., 1991, Methods in Enzym. 203:46-121). In a further embodiment, the linker can span the distance between its points of fusion to each of the variable domains (e.g., 3.5 nm) to minimize distortion of the native Fv conformation. In such an embodiment, the linker is a polypeptide of at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, or greater. In a further embodiment, the linker should not cause a steric interference with the VH and VL domains of the combining site. In such an embodiment, the linker is 35 amino acids or less, 30 amino acids or less, or 25 amino acids or less. Thus, in a most preferred embodiment, the linker is between 15-25 amino acid residues in length. In a further embodiment, the linker is hydrophilic and sufficiently flexible such that the VH and VL domains can adopt the conformation necessary to detect antigen. Intrabodies can be generated with different linker sequences inserted between identical VH and VL domains. A linker with the appropriate properties for a particular pair of VH and VL domains can be determined empirically by assess the degree of antigen binding for each. Examples of linkers include, but are not limited to, those sequences disclosed in Table 1A.

TABLE 1A Sequence (SEQ ID NO: 5) (Gly Gly Gly Gly Ser)3 (SEQ ID NO: 6) Glu Ser Gly Arg Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser (SEQ ID NO: 7) Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr (SEQ ID NO: 8) Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr Gln (SEQ ID NO: 9) Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys  Val Asp (SEQ ID NO: 10) Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Glu Gly Lys Gly (SEQ ID NO: 11) Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser Leu Asp (SEQ ID NO: 12) Glu Ser Gly Ser Val Ser Ser Glu Glu Leu Ala Phe Arg Ser Leu Asp

In one embodiment, intrabodies are expressed in the cytoplasm. In other embodiments, the intrabodies are localized to various intracellular locations. In such embodiments, specific localization sequences can be attached to the intranucleotide polypepetide to direct the intrabody to a specific location. Intrabodies can be localized, for example, to the following intracellular locations: endoplasmic reticulum (Munro et al., 1987, Cell 48:899-907; Hangejorden et al., 1991, J. Biol. Chem. 266:6015); nucleus (Lanford et al., 1986, Cell 46:575; Stanton et al.,1986, PNAS 83:1772; Harlow et al., 1985, Mol. Cell Biol. 5:1605); nucleolar region (Seomi et al., 1990, J. Virology 64:1803; Kubota et al., 1989, Biochem. Biophys. Res. Comm. 162:963; Siomi et al., 1998, Cell 55:197); endosomal compartment (Bakke et al., 1990, Cell 63:707-716); mitochondrial matrix (Pugsley, A. P., 1989, “Protein Targeting”, Academic Press, Inc.); Golgi apparatus (Tang et al., 1992, J. Bio. Chem. 267:10122-6); liposomes (Letourneur et al., 1992, Cell 69:1183); and plasma membrane (Marchildon et al., 1984, PNAS 81:7679-82; Henderson et al., 1987, PNAS 89:339-43; Rhee et al., 1987, J. Virol. 61:1045-53; Schultz et al., 1984, J. Virol. 133:431-7; Ootsuyama et al., 1985, Jpn. J. Can. Res. 76:1132-5; Ratner et al., 1985, Nature 313:277-84). Examples of localization signals include, but are not limited to, those sequences disclosed in Table 2A.

TABLE 2A Localization Sequence (SEQ ID NO: 13) endoplasmic reticulum Lys Asp Glu Leu (SEQ ID NO: 14) endoplasmic reticulum Asp Asp Glu Leu (SEQ ID NO: 15) endoplasmic reticulum Asp Glu Glu Leu (SEQ ID NO: 16) endoplasmic reticulum Gln Glu Asp Leu (SEQ ID NO: 17) endoplasmic reticulum Arg Asp Glu Leu (SEQ ID NO: 18) nucleus Pro Lys Lys Lys Arg Lys Val (SEQ ID NO: 19) nucleus Pro Gln Lys Lys Ile Lys Ser (SEQ ID NO: 20) nucleus Gln Pro Lys Lys Pro (SEQ ID NO: 21) nucleus Arg Lys Lys Arg (SEQ ID NO: 22) nucleolar region Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala His Gln (SEQ ID NO: 23) nucleolar region Arg Gln Ala Arg Arg Asn Arg Arg Arg Arg Trp Arg Glu Arg Gln Arg (SEQ ID NO: 24) nucleolar region Met Pro Leu Thr Arg Arg Arg Pro Ala Ala Ser Gln Ala Leu Ala Pro Pro Thr Pro (SEQ ID NO: 25) endosomal Met Asp Asp Gln Arg Asp Leu Ile (SEQ ID NO: 26) compartment Ser Asn Asn Glu Gln Leu Pro (SEQ ID NO: 27) mitochondrial matrix Met Leu Phe Asn Leu Arg Xaa Xaa Leu Asn Asn Ala Ala Phe Arg His Gly His Asn Phe Met Val Arg Asn Phe Arg Cys Gly Gln Pro Leu Xaa (SEQ ID NO: 28) plasma membrane GCVCSSNP (SEQ ID NO: 29) plasma membrane GQTVTTPL (SEQ ID NO: 30) plasma membrane GQELSQHE (SEQ ID NO: 31) plasma membrane GNSPSYNP (SEQ ID NO: 32) plasma membrane GVSGSKGQ (SEQ ID NO: 33) plasma membrane GQTITTPL (SEQ ID NO: 34) plasma membrane GQTLTTPL (SEQ ID NO: 35) plasma membrane GQIFSRSA (SEQ ID NO: 36) plasma membrane GQIHGLSP (SEQ ID NO: 37) plasma membrane GARASVLS (SEQ ID NO: 38) plasma membrane GCTLSAEE

VH and VL domains are made up of the immunoglobulin domains that generally have a conserved structural disulfide bond. In embodiments where the intrabodies are expressed in a reducing environment (e.g., the cytoplasm), such a structural feature cannot exist. Mutations can be made to the intrabody polypeptide sequence to compensate for the decreased stability of the immunoglobulin structure resulting from the absence of disulfide bond formation. In one embodiment, the VH and/or VL domains of the intrabodies contain one or more point mutations such that their expression is stabilized in reducing environments (see Steipe et al., 1994, J. Mol. Biol. 240:188-92; Wirtz and Steipe, 1999, Protein Science 8:2245-50; Ohage and Steipe, 1999, J. Mol. Biol. 291:1119-28; Ohage et al., 1999, J. Mol Biol. 291:1129-34).

Methods for Producing Antibodies

The antibodies to be used with the methods of the invention or fragments thereof can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.

Polyclonal antibodies to vaccine component of the invention can be produced by various procedures well known in the art. For example, an antigen according to the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, bacterial ghosts, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art. In the case of ghosts the antigens are preferably coupled to the ghost, for instance through protein anchors, of which the AcmA protein anchor is especially preferred.

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981). The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. Ascites fluid, which generally contains high levels of antibodies; can be generated by immunizing mice with positive hybridoma clones.

In certain embodiments, a method of generating monoclonal antibodies comprises culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind said antigen.

Antibody fragments which recognize the specific antigens of the invention may be generated by any technique known to those of skill in the art. For example, Fab and F(ab′)2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain. Further, the antibodies to be used with the present invention can also be generated using various phage display methods known in the art.

In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of lymphoid tissues). The DNA encoding the VH and VL domains are recombined together with an scFv linker by PCR and cloned into a phagemid vector (e.g., p CANTAB 6 or pComb 3 HSS). The vector is electroporated in E. coli and the E. coli is infected with helper phage. Phage used in these methods are typically filamentous phage including fd and M13 and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene VIII. Phage expressing an antigen binding domain that binds to an antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177-186; Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al., 1994, Advances in Immunology 57:191-280; PCT application No. PCT/GB91/O1 134; PCT publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401, and WO97/13844; and U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108.

As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e g., as described below. Techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication No. WO 92/22324; Mullinax et al., 1992, BioTechniques 12(6):864-869; Sawai et al., 1995, AJRI 34:26-34; and Better et al., 1988, Science 240:1041-1043.

To generate whole antibodies, PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences in scFv clones. Utilizing cloning techniques known to those of skill in the art, the PCR amplified VH domains can be cloned into vectors expressing a VH constant region, e.g., the human gamma 4 constant region, and the PCR amplified VL domains can be cloned into vectors expressing a VL constant region, e.g., human kappa or lamba constant regions. Preferably, the vectors for expressing the VH or VL domains comprise an EF-1α promoter, a secretion signal, a cloning site for the variable domain, constant domains, and a selection marker such as neomycin. The VH and VL domains may also cloned into one vector expressing the necessary constant regions. The heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques known to those of skill in the art.

For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use human or chimeric antibodies. Completely human antibodies are particularly desirable for therapeutic treatment of human subjects. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences or synthetic sequences homologous to human immunoglobulin sequences. See also U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO 96/33735, and WO 91/10741.

Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then be bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publication Nos. WO 98/24893, WO 96/34096, and WO 96/33735; and U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598. In addition, companies such as Medarex, Inc. (Princeton, N.J), Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

A chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such as antibodies having a variable region derived from a non-human (e.g., murine) antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567, and 4,816,397. Chimeric antibodies comprising one or more CDRs (complementarity determining regions) from human species and framework regions from a non-human immunoglobulin molecule can be produced using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska et al., 1994, PNAS 91:969-973), and chain shuffling (U.S. Pat. No. 5,565,332). In a preferred embodiment, antibodies comprise one or more CDRs and human framework regions. Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature 332:323.)

Polynucleotides encoding antibodies to be used with the invention may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. Such a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242).

Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.

In a specific embodiment, one or more of the CDRs is inserted within framework regions using routine recombinant DNA techniques. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., 1998, J. Mol. Biol. 278: 457-479 for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds to a vaccine component of the invention. In certain embodiments, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.

Recombinant expression of an antibody to be used with the methods of the invention, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a portion thereof or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once such a polynucleotide has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention.

Antibodies according to the invention can not only be used for passive vaccination, but can also be used for diagnostic purposes and in assay systems to detect presence of bacteria and/or the antigens to which they are directed. Such a diagnosis is useful (for instance) to test material (such as food, pharmaceutical compositions or air filters) for the presence of bacteria. It is also possible to develop a test system which can detect the antibodies themselves to check for the presence or to calculate the titer of antibodies in the blood of subjects that have been immunized with a vaccine according to the invention.

A person skilled in the art is well able to generate analogous compounds of the opsonophagocytosis inducing proteins of the invention. This can for instance be done through screening of a peptide library and testing to binding to one or more of the antibodies described above. Such an analogue has essentially the same biological properties of said protein in kind, i.e. it is able to generate a similar immunological response, not necessarily in amount.

It will also be possible genetically engineer analogous compounds starting from the sequences provided in this specification. Such analogous compounds will have a sequence identity of more than 50% compared to the sequences given in the specification. Sequence identity may be defined and determined by the TBLASTN or TBLASTP program for nucleic acid or amino acid sequences, respectively, of Altschul et al. (1990), which is in standard use in the art, or, and this may be preferred, the standard program BestFit, which is part of the Wisconsin Package, Version 8, September 1994, (Genetics Computer Group, 575 Science Drive, Madison, Wis., USA, Wisconsin 53711). Sequence identity on nucleotide sequences can be calculated by using the BLASTN computer program (which is publicly available, for instance through the National Center for Biotechnological Information, accessible via the internet on http://www.ncbi.nlm.nih.gov/) using the default settings of 11 for wordlength (W), 10 for expectation (E), 5 as reward score for a pair of matching residues (M), −4 as penalty score for mismatches (N) and a cutoff of 100. Sequence identity may be at the nucleotide sequence and/or encoded amino acid sequence level. Preferably the nucleic acid and/or amino acid sequence shares at least 50%, or 60% sequence identity, most preferably at least about 70%, or 80% or 90% sequence identity with the sequences of the specification as provided hereunder.

It is also possible to search for orthologous proteins, i.e. proteins from other organisms, be it closely related or not to Strepococcus pneumoniae D39 and/or Strepococcus pneumoniae TIGR4, such as other Strepococcus pneumoniae strains, or other Strepococcus species, or even Staphylococcus, Moraxella, Neisseria and other bacteria, or even non-bacterial organisms. Orthologous proteins in this regard means proteins from other species which proteins have the same common ancestry, but are different in sequence as a result of (spontaneous) mutations. These sequences can easily been found, for instance by comparing the sequences of the invention to other sequences available in sequence databases such as GenBank or by isolating orthologous proteins from other organisms by PCR mediated amplification methods in which (degenerated) primers are designed based on the sequences of the specification. Such methods are known to the person skilled in the art.

The proteins obtained by the above described methods should further be tested with respect to their opsonophagocytosis inducing properties. Any protein obtained through the above discussed methods which appears to be opsonophagocytosis inducing is considered to be a protein of the invention and is considered to be suitable for inclusion into the vaccines of the invention. Also parts of these proteins which have retained the functional characteristic of being able to induce opsonophagocytosis are considered to be comprised in the present invention.

Opsonophagocytosis inducing proteins as vaccine components can also be combined with other opsonophagocytosis inducing proteins and/or other proteins to result in a better vaccine. This patent application discloses the use of SlrA, IgA1 proteinase and PsaA and the combination of PpmA and/or other proteins with said proteins. Preferably, a vaccine according to the invention comprises two of more opsonophagocytosis inducing proteins, wherein the combination of PpmA and SlrA, whether or not in the presence of further antigens, is most preferred.

The invention is further explained in the examples.

EXAMPLE 1

Correlation of Antibody Titers Against Strepococcus pneumoniae Surface Proteins with Occurrence of first Acute Otitis Media.

A Cross Sectional Population Survey in Finland.

As is shown in FIGS. 1-5 in people with first occurrence of otitis media, the IgG titers against PpmA, SlrA, and IgA1 proteinase, are all lower than the titers against alpha-enolase of Strepococcus pneumoniae. Because of the absence of this correlation of antibody titer and protection, SlrA and IgA1 proteinase were considered to have no added value for protective immunity, and hence would not be selected for use as a vaccine component.

EXAMPLE 2

Immune Protective Potential of Strepococcus pneumoniae Surface proteins

To determine the protective potential of Strepococcus pneumoniae surface proteins, both active and passive immunization/challenge experiments were performed.

Methods

Mouse Strains

Immunisation was carried out on MF1 outbred female mice, purchased from Harlan Olac, Bicester, UK All mice were barrier reared and specified pathogen free. The active immunisation protocol started with 6 week old mice (25-30g) and passive immunisation with 9 week old mice (30-35g).

Bacterial Strains

Strepococcus pneumoniae D39, serotype 2, has been obtained from the National Collection of Type Cultures (NCTC 7466; Central Public Health Laboratory, London). Strepococcus pneumoniae TIGR4, serotype 4, has been obtained from The Institute for Genome Research. Bacteria are grown on Blood Agar Base Number 2 (Oxoid, Basingstoke, UK) plus 5% (vol/vol) defibrinated horse blood (BAB). Stock cultures are prepared by inoculating a sweep of 4-5 colonies into 25ml Brain-Heart Infusion broth (BHI, Oxoid). Cultures are grown statically at 37° C. overnight before 1 ml aliquots containing 15% sterile glycerol are frozen at −70° C. until use. Strain validation is carried out by confirming sensitivity to the antibiotic Optochin (Difco, Detroit, USA). Serotype verification is performed by checking for the presence of type specific polysaccharide capsule-using anti-type 2 or 4 capsule antiserum (Statens Seruminstitut, Copenhagen, Denmark).

For counting of CFU, serial dilutions of samples are prepared by the addition of 20 μl to 180 μl sterile PBS in sterile round-bottomed 96 well microtitre plates (Life Technologies, Paisley, UK). 20 μl volumes of each dilution are spotted in triplicate on 2 separate BAB plates. The following equation is used to calculate the number of colony forming units per ml of sample: CFU per ml=Mean number of colonies per sector×50×Dilution Factor. Preparation of Inoculums

100μl of sterile PBS containing approximately 10⁵ CFU Strepococcus pneumoniae were injected into the peritoneal cavity of mice with an insulin syringe (F. Baker Scientific, Runcorn, UK). On development of lethargy, mice were killed by cervical dislocation, the chest cavity carefully opened up and a section of ribcage removed in order to provide access to the heart. A 23 gauge needle was inserted into the right ventricle and blood slowly collected into the attached syringe. 50 μl of infected blood was used to grow passaged bacteria in 10 ml BHI statically overnight at 37° C. Bacteria were harvested by centrifugation at 3,000 rpm for 10 min (Heraeus medifuge), resuspended in fresh BHI containing 20% v/v FCS and then incubated at 37° C. until they reached mid-log phase (as judged by optical density). 1 ml aliquots of the suspension were stored at −70° C., until required. After 24h at −70° C. viability of the suspension was measured by viable counting and optochin sensitivity was confirmed. Viability was unaffected by storage at −70° C. for at least 3 months.

Intranasal Infection

Aliquots of standard inoculum were rapidly thawed. A 900 μl sample was pelleted by centrifugation at 13,000 rpm (Eppendorf centrifuge 5417 C.) and resuspended in 9001 μl sterile PBS. Bacteria were diluted in sterile PBS to give 1×10⁶ CFU/50 μl and this confirmed by viable counting (as described previously). Mice to be infected were lightly anesthetized with 2.5% v/v isoflourane over oxygen (1.5 L/min) with NO₂ (1 L/min), administered using a calibrated vaporizer and anaesthesia confirmed by observing no pinch reflex reaction. Once anesthetized the animals were held with the nose held upright, and pneumococci were introduced intranasally by adding a series of small droplets of the 50 μL inoculum in to the nostril for the mice to involuntarily inhale. After inoculation the mice were laid on their backs until recovery and the viable count of the inoculum was re-determined. Mice were monitored frequently (at least 3 times a day) during the following 14 days for signs of infection. On reaching the pre-determined endpoint (moribund) mice were killed by cervical dislocation and the survival time noted.

Passive Immunization

Serum was complement inactivated by incubation at 56° C. for 30 min and was used at a pre-determined dilution. 24 h and 1 h prior to infection, mice were vaccinated by intraperitoneal injection of 200 μl serum. Mice were then infected as previously described. Bacteria were opsonized by incubation in the diluted serum at 37° C. for 1 hr prior to infection. Mice were monitored frequently (at least 3 times a day) during the following 14 days for signs of infection. On reaching the pre-determined endpoint (moribund) mice were sacrificed by cervical dislocation and the survival time noted.

Active Immunization

Mice were randomly arranged in groups of 6 and a blood sample was removed from the tail vein prior 24 h prior to initial vaccination. Vaccines were prepared in a 1:1 ratio in either Freunds complete adjuvant or incomplete adjuvant (Sigma) or Hunter's TiterMax (CytRx Corp., Norcross, Ga.) or Quill A (Accurate Chemical Scientific Corporation, Westbury, N.Y. 11590) as per manufacturers instructions. Normally 50 μg of protein was administered per mouse. 100 μl of vaccine was injected subcutaneously per mouse. Nine days later a blood sample was removed from half of the mice from a tail vein. By staggering this bleed and the next we could bleed all mice prior to vaccination and prior to infection but still monitor immune response during vaccination protocol: this kept us in line with the restrictions on the total number of bleeds/mouse (3) and the minimum time interval between bleeds (14 days). 24 h after sampling blood, vaccination was repeated as previously but if using Freunds, FIA (Freunds incomplete adjuvant) was used. 9 days after boost, a blood sample was removed from those mice not bled before last boost. 24 h later vaccination was repeated as previously. 28 days later a blood sample was removed from the tail vein of all mice. 24 h later mice were infected as previously described with approximately 10⁶ CFU Strepococcus pneumoniae D39 or TIGR4. Mice were monitored at least 3 times daily for signs of infection. On reaching the pre-determined endpoint (moribund) mice were sacrificed by cervical dislocation.

Preparation of Mouse Virulent Inoculum

During the course of these experiments, a maximum of 25 mice were used for the production of mouse virulent inocula. The number of times that inocula must be prepared/passaged depended on the virulence and viability of the organisms after prolonged freezing at −80° C.

Infection Experiments

In order to optimize the pneumonia infection model in our laboratory, i.e. to establish the optimal number of organisms that will produce consistent pneumonia with the longest incubation period, we infected groups of 4 mice with 5 different dilutions of organisms from 10³, to 10⁷ cfu). We optimized the infection dose required for two heterogeneous strains of Strepococcus pneumoniae (D39 and TIGR4). These strains differ in their virulence and antigen characteristics. (4×5×2=40 mice).

Passive Immunization

For passive immunization with rabbit polyclonal anti-sera, the following sera were tested.

Pre-immune serum PpmA

Polyclonal serum PpmA

6 mice per group; challenged with wild virulent strain of Strepococcus pneumoniae and with a strain D39 deleted for PpmA.

TABLE 1 Time Schedule Active immunization Week day Function Housing 0-2 Arrival and Normal/group acclimatization of mice (4 week old) 2  0 Collect pre-immune Normal/group serum 2  1 Primary vaccination Normal/group 3 10 2^(nd) serum collection Normal/group 3 11 1^(st) boost immunization Normal/group 4 20 3^(rd) serum collection Normal/group 4 21 2^(nd) boost Normal/group immunization 8 49 4^(th) serum collection Normal/group 9 50 Inoculation Small cage, filter top  9-11 50-64 Survival time - serum Small cage, collection - filter top pathology 11  64 Experiment end - Small cage, serum filter top collection - pathology

TABLE 2 Time Schedule Passive immunization Week day Function Housing 0-2 Arrival and acclimatization of mice (7 Normal/group week old) 2 0 1^(st) intraperitoneal serum injection Normal/group 2 1 2^(nd) intraperitoneal serum injection Normal/group 2 1 Inoculation 1 hour later Small cage, filter top 2-4 1-14 Survival time - serum collection - Small cage, pathology filter top 4 14  Experiment end - serum collection - Small cage, pathology filter top

TABLE 3 Time Schedule for preparation of inoculum Week day Function Housing 0-2 Arrival and acclimatization of mice Normal/group (7 week old) 2 0 Intraparietal inoculation with 10⁵ cfu Small cage, filter top 2-3 0-7 Onset of lethargy - terminate - collect Small cage, blood filter top 3 7 Experiment end Small cage, filter top Results:

Active immunization of CD-1 mice with Strepococcus pneumoniae surface protein and infection with a wild type virulent strain of Strepococcus pneumoniae.

Immune Protective Potentials of SlrA

SlrA elicited a good protective immune response in the mice, as is shown in table 4 and FIG. 6

TABLE 4 Survival time in hours after active immunization with SlrA n = 12 Control SlrA 49.67 53.16 52.25 53.16 47.34 60.33 44.67 200 47 58.83 43.17 146.33 200 53.42 45.66 200 200 50.17 52.5 47.83 52.5 57.67 49.83 47.58

Statistically, a significant difference was found between the vaccinated and the control group concerning the survival time of mice after challenge with virulent Strepococcus pneumoniae D39.

Mann Whitney test P value  0.0175 Gaussian Approximation P value. P value summary Medians are significantly different (P < 0.05) One-tailed P value Sum of ranks in column A, B  113, 187 Mann-Whitney U  35.00 Immune Protective Potentials of IgA1 Proteinase

IgA1 proteinase elicited a good protective immune response in the mice, as is shown in table 5 and FIG. 7

TABLE 5 Survival time in hours after active immunization with IgA1 proteinase (n = 10) Control IgA1 >250 47.5 >250 49.75 42.13 169.5 58.33 70.75 47.25 168.5 43.13 >250 50.33 164.5 48.33 >250 53.33 58.42 >250

Statistically, a significant difference was found between the vaccinated and the control group concerning the survival time of mice after challenge with virulent Strepococcus pneumoniae D39.

Mann Whitney test P value  0.0474 Gaussian Approximation of P-value P value summary Medians are significantly different (P < 0.05) One-tailed P value. Sum of ranks in column A, B  69, 121 Mann-Whitney U  24.00 Results are shown in FIG. 7. Immune Protective Potentials of PsaA

PsaA elicited a good protective immune response in the mice, as is shown in FIG. 8.

Statistically, a significant difference was found between the vaccinated and the control group concerning the survival time after challenge with virulent Strepococcus pneumoniae D39.

Mann Whitney test P value  0.0409 Gaussian Approximation of P value. P value summary Medians are significantly different (P < 0.05) Sum of ranks in column A, B  195.5, 300.5 Mann-Whitney U  75.50 Combination Experiments Vaccinating with at Least one Surface Protein.

Vaccinating with two antigenic proteins resulted in a better survival time in mice after challenge with virulent Strepococcus pneumoniae D39 as is shown in table 6, and in FIG. 9.

TABLE 6 Survival time in hours after active immunization with PpmA and SlrA. N = 12 PpmA SlrA PpmA + SlrA 53 53.16 56.25 50.34 53.16 56.25 43.67 60.33 50.58 56.17 >200 56.25 56.47 58.83 56.75 53.5 146.33 56.75 >200 53.42 72.17 57.58 >200 >200 53.58 50.17 54.67 58.58 47.83 72.17 47.58 57.67 54.75 47.58 47.58 56.67

PpmA vs PpmA + SlrA Mann Whitney test P value  0.0416 Gaussian Approximation of P value. P value summary Medians are significantly different (P < 0.05) One-tailed P value. Sum of ranks in column A, C  119.5, 180.5 Mann-Whitney U  41.50

SlrA vs PpmA + SlrA Mann Whitney test P value  0.3325 Gaussian Approximation of P value. P value Summary ns Medians are not significantly different. (P < 0.05) One-tailed P value Sum of ranks in column B, C  142, 158 Mann-Whitney U  64.00

Statistically, there was a significant difference between the PpmA and SlrA combined group and the PpmA alone. This shows that protective immunity as a result of vaccination with more than one protein or part thereof is superior to the immunity based on vaccination with only one antigen.

Passive Immunization of CD-1 mice with Anti-PpmA Serum and infection with a PpmA-ve Control Strain

Control—mice were vaccinated (2×200 μl) with rabbit-pre immune serum (PIS)

Experiment—mice vaccinated with (2×200 μl) with polyclonal rabbit hyperimmune anti-rPpmA serum (APS).

TABLE 7 Survival time in hours after passive immunization with PpmA- antibodies (APS) or with normal pre-immune serum (PIS) PIS D39+ APS D39+ PIS PpmA- APS PpmA- 59.00 113.67 47.17 118.33 59.00 40.67 51.67 118.33 65.00 63.17 59.67 65.82 65.00 59.17 48.67 65.82 59.00 90.17 90.67 59.82 46.25 59.17 38.17 72.82 59.00 59.17 >200 46.82 46.50 63.17 38.5 51.82 46.25 >200 >200 65.82 46.75 48.17 61 46.82 Passive Immunization Experiment Mice Infected Intranasally with Strepococcus pneumoniae D39

Mann Whitney test P value  0.0376 Exact P value P value summary * Medians significantly different (P < 0.05) One-tailed P value Sum of ranks in column A, B  81, 129 Mann-Whitney U  26.00 Passive immunization Experiment. Mice Infected Intranasally with Strepococcus pneumoniae D39 ppmA-mutant

Mann Whitney test P value  0.2644 Exact P value P value summary ns Medians are not significantly different (P < 0.05) One-tailed P value Sum of ranks in column C, D  96, 114 Mann-Whitney U  41.00

The results in table 7 and in FIG. 10 clearly show the protective effect of passive immunization with anti PpmA antibodies against a challenge infection with virulent streptococcal strain D39. They also show that the protection is much less against a streptococcal strain that is deleted for PpmA. The ppmA-ve D39 strain has been previously shown to be less virulent than D39.

TABLE 8 Survival time in hours after passive immunization with PpmA in MF-1 mice Treatment Survival times (h) Normal rabbit serum (NRS) 41, 48, 48, 65, 72 Bacteria opsonised in NRS 48, 48, 65, 72, 89 Anti-PpmA anti-serum 71, 71, 113.5, 113.5, 137, 336, 336, 336, 336, 336 Bacteria opsonised in anti-PpmA 89, 143, 336, 336, 336

The results in table 8 and in FIG. 11 clearly show the protection against challenge infection after a passive immunization.

EXAMPLE 3

In Silico Search

The nucleotide sequence of PpmA was determined to search databases

The gene for PpmA was amplified by PCR from Strepococcus pneumoniae D39 with the primers 5′ CCATGGCTAGCCACCATCACCATCACCATTCGAAAGGGTCAGAAGGTGC 3′ (SEQ ID NO: 60) and 5′ TCATGGATCCGGACTATTCGTTTGATGTAC3′ (SEQ ID NO: 61) which incorporate flanking NheI and BamHI restriction sites, and a N-terminal HiS ⁶ tag. The amplified DNA was cloned into a pET11a expression vector (Stratagene, LaJolla, Calif.) and electrotransformed into E. coli BL21(DE3). The recombinant protein was purified by Ni ⁺ affinity chromatography with the HisTrap Kit (Amersham Pharmacia) according to the manufacturers recommendations.

The purified recombinant protein was dialysed against 10 mM HEPES buffer, pH 7.5, freeze dried, and stored at −20° C.

PpmA ppmA nucleotide sequence Streptococcus pneumoniae D39 (SEQ ID NO: 39) ATGAAGAAAAAATTATTGGCAGGTGCCATCACACTATTATCAGTAGCAAC TTTAGCAGCTTGTTCGAAAGGGTCAGAAGGTGCAGACCTTATCAGCATGA AAGGGGATGTCATTACAGAACATCAATTTTATGAGCAAGTGAAAAGCAAC CCTTCAGCCCAACAAGTCTTGTTAAATATGACCATCCAAAAAGTTTTTGA AAAACAATATGGCTCAGAGCTTGATGATAAAGAGGTTGATGATACTATTG CCGAAGAAAAAAAACAATATGGCGAAAACTACCAACGTGTCTTGTCACAA GCAGGTATGACTCTTGAAACACGTAAAGCTCAAATTCGTACAAGTAAATT AGTTGAGTTGGCAGTTAAGAAGGTAGCAGAAGCTGAATTGACAGATGAAG CCTATAAGAAAGCCTTTGATGAGTACACTCCAGATGTAACGGCTCAAATC ATCCGTCTTAATAATGAAGATAAGGCCAAAGAAGTTCTCGAAAAAGCCAA GGCAGAAGGTGCTGATTTTGCTCAATTAGCCAAAGATAATTCAACTGATG AAAAAACAAAAGAAAATGGTGGAGAAATTACCTTTGATTCTGCTTCAACA GAAGTACCTGAGCAAGTCAAAAAAGCCGCTTTCGCTTTAGATGTGGATGG TGTTTCTGATGTGATTACAGCAACTGGCACACAAGCCTACAGTAGCCAAT ATTACATTGTAAAACTCACTAAGAAAACAGAAAAATCATCTAATATTGAT GACTACAAAGAAAAATTAAAAACTGTTATCTTGACTCAAAAACAAAATGA TTCAACATTTGTTCAAAGCATTATCGGAAAAGAATTGCAAGCAGCCAATA TCAAGGTTAAGGACCAAGCCTTCCAAAATATCTTTACCCAATATATCGGT GGTGGAGATTCAAGCTCAAGCAGTAGTACATCAAACGAATAG PpmA amino acid sequence (SEQ ID NO: 40) MKKKLLAGAITLLSVATLAACSKGSEGADLISMKGDVITEHQFYEQVKSN PSAQQVLLNMTIQKVFEKQYGSELDDKEVDDTIAEEKKQYGENYQRVLSQ AGMTLETRKAQIRTSKLVELAVKKVAEAELTDEAYKKAFDEYTPDVTAQI IRLNNEDKAKEVLEKAKAEGADFAQLAKDNSTDEKTKENGGEITFDSAST EVPEQVKKAAFALDVDGVSDVITATGTQAYSSQYYIVKLTKKTEKSSNID DYKEKLKTVILTQKQNDSTFVQSIIGKELQAANIKVKDQAFQNIFTQYIG GGDSSSSSSTSNE* Recombinant His tagged PpmA (SEQ ID NO: 41) MAHHHHHHSKGSEGADLISMKGDVITEHQFYEQVKSNPSAQQVLLNMTIQ KVFEKQYGSELDDKEVDDTIAEEKKQYGENYQRVLSQAGMTLETRKAQIR TSKLVELAVKKVAEAELTDEAYKKAFDEYTPDVTAQIIRLNNEDKAKEVL EKAKAEGADFAQLAKDNSTDEKTKENGGEITFDSASTEVPEQVKKAAFAL DVDGVSDVITATGTQAYSSQYYIVKLTKKTEKSSNIDDYKEKLKTVILTQ KQNDSTFVQSIIGKELQAANIKVKDQAFQNIFTQYIGGGDSSSSSSTSNE SlrA

The gene for the active peptide of SirA was amplified by PCR from Strepococcus pneumoniae D39 with the primers 5′TTTACTGCATATGCACCATCACCATCACCATAGCAGCGTCCAACGCAGT3′ (SEQ ID NO: 42) and 5′CATTAGGATCCAATCGCTGGGGAAGTG3′ (SEQ ID NO: 43) which incorporate flanking NdeI and BamHI restriction sites, and a N-terminal His ⁶ tag. The amplified DNA was cloned into a pET11a expression vector (Stratagene, LaJolla, Calif.) and electrotransformed into E. coli BL21(DE3). The recombinant protein was purified by Ni ⁺ affinity chromatography with the HisTrap Kit (Amersham Pharmacia) according to the manufacturers recommendations. The purified recombinant protein was dialysed against 10 mM HEPES buffer, pH7.5, freeze dried, and stored at −20° C.

SlrA slrA nucleotide sequence Streptococcus pneumoniae D39 (SEQ ID NO: 44) ATGAAAAAACTAGCAACCCTTCTTTTACTGTCTACTGTAGCCCTAGCTGG GTGTAGCAGCGTCCAACGCAGTCTGCGTGGTGATGATTATGTTGATTCCA GTCTTGCTGCTGAAGAAAGTTCCAAAGTAGCTGCCCAATCTGCCAAGGAG TTAAACGATGCTTTAACAAACGAAAACGCCAATTTCCCACAACTATCTAA GGAAGTTGCTGAAGATGAAGCCGAAGTGATTTTCCACACAAGCCAAGGTG ATATTCGCATTAAACTCTTCCCTAAACTCGCTCCTCTAGCGGTTGAAAAT TTCCTCACTCACGCCAAAGAAGGCTACTATAACGGTATTACCTTCCACCG TGTCATCGATGGCTTTATGGTCCAAACTGGAGATCCAAAAGGGGACGGTA CAGGTGGTCAGTCCATCTGGCATGACAAGGATAAGACTAAAGACAAAGGA ACTGGTTTCAAGAACGAGATTACTCCTTATTTGTATAACATCCGTGGTGC TCTTGCTATGGCTAATACTGGTCAACCAAACACCAATGGCAGCCAGTTCT TCATCAACCAAAACTCTACAGATACCTCTTCTAAACTCCCTACAAGCAAG TATCCACAGAAAATTATTGAAGCCTACAAAGAAGGTGGAAACCCTAGTCT AGATGGCAAACACCCAGTCTTTGGTCAAGTGATTGACGGTATGGATGTTG TGGATAAGATTGCTAAGGCCGAAAAAGATGAAAAAGACAAGCCAACTACT GCTATCACAATCGACAGCATCGAAGTGGTGAAAGACTACGATTTTAAATC TTAA SlrA amino acid sequence (SEQ ID NO: 45) MKKLATLLLLSTVALAGCSSVQRSLRGDDYVDSSLAAEESSKVAAQSAKE LNDALTNENANFPQLSKEVAEDEAEVIFHTSQGDIRIKLFPKLLAPLAVE NFLTHAKEGYYNGITFHRVIDGFMVQTGDPKGDGTGGQSIWHDKDKTKDK GTGFKNEITPYLYNIRGALAMANTGQPNTNGSQFFINQNSTDTSSKLPTS KYPQKILEAYKEGGNPSLDGKHPVFGQVIDGMDVVDKIAKAEKDEKDKPT TAITIDSIEVVKDYDFKS Recombinant His tagged SlrA (SEQ ID NO: 46) MAHHHHHHSSVQRSLRGDDYVDSSLAAEESSKVAAQSAKELNDALTNENA NFPQLSKEVAEDEAEVIFHTSQGDIRIKLFPKLAPLAVENFLTHAKEGYY NGITFHRVIDGFMVQTGDPKGDGTGGQSIWHDKDKTKDKGTGFKNEITPY LYNIRGALAMANTGQPNTNGSQFFINQNSTDTSSKLPTSKYPQKIIEAYK EGGNPSLDGKHPVFGQVIDGMDVVDKIAKAEKDEKDKPTTAITIDSIEVV KDYDFKS Expression and Purification of His⁶rIgA1 Proteinase

A 3948 bp gene fragment which encodes amino acids 671-1987 of the IgA1 proteinase was amplified by PCR from Strepococcus pneumoniae TIGR4 with the primers 5′CAACATCACATATGGGACAAACAGAACCAGAG3′ (SEQ ID NO: 47) and 5′ACTTAGATCTTAATGATGGTGGTGATGATGGGCTTTAAAGATTGCTCTC3′ (SEQ ID NO: 48) which incorporate flanking Ndel and BglII restriction sites, and a C-terminal His ⁶ tag. The amplified DNA was cloned into a pET11a expression vector (Stratagene, LaJolla, Calif.) and electrotransformed into E. coli BL21(DE3).

Inoculate 200 ml LB broth with 50 μl/ml ampicillin and grow at 37° C. overnight.

Transfer 20-30 ml per liter of the overnight culture to prewarmed LB with 50 μl/ml ampicillin and grow to an OD₆₀₀ between 0.6 and 1.0 (2-3 hours).

Expression is induced by the addition of 1 mm IPTG (10 ml of a 100 mM stock/liter), and the cells were grown for a further 2-3hours.

Cells were harvested (10 min @ 3000 rpm) and resuspended in ice cold 20 mM sodium phosphate buffer pH7.4 with 8M urea (15 ml of buffer/liter of culture).

The cells are lysed by sonication 3×30 seconds on ice (1 min intervals to allow cooling).

Place lysate on a rotating shaker at 4° C. for 20 min (to dissolve inclusion bodies).

The crude lysate is cleared by centrifugation (15,000 rpm for 30 min in a JA20 rotor).

Place the cleared supernatant in a pre-boiled dialysis bag, and dialyze overnight against 2 L ice cold 20 mM sodium phosphate buffer pH7.4 with 1 mM DTT.

Add ammonium sulfate to the dialyses supernatant to 20% saturation (0.123 g/ml), and stir at 4° C. for 20 min.

Spin precipitate at 10,000 rpm for 15 min in a JA20 rotor at 4° C. (IgA1 proteinase will be recovered from the protein pellet).

Redisolve protein pellet (5 ml/liter culture) in start buffer (2 mM sodium phosphate buffer pH7.4, 0.5M NaCl ,10 mM imidazole 8M urea).

Filter through a 0.45 μm filter.

Load 1 ml column with Ni⁺ and equilibrate it with start buffer with urea.

Pump the supernatant (15 ml) through a Ni⁺ loaded 1 ml HisTrap column (Amersham Pharmacia).

Wash with 10 ml wash buffer (20 mM sodium phosphate buffer pH7.4, 0.5M NaCl , 20 mM imidazole 8M urea).

Elute in 1 ml fractions with elution (20 mM sodium phosphate buffer pH7.4; 0.5M NaCl; 400 mM imidazole; 8M urea).

The second fraction should contain the IgA1 proteinase.

The 2^(nd) fraction is then dialyzed overnight against 2 L 10 mM Hepes buffer pH7.4.

Without the Urea, the protein will re-precipitate. Recover the protein by centrifugation, Freeze dry and store at −20° C.

Protein can be resolubilized in 8M urea and then diluted in PBS etc. It will remain soluble at high concentrations in 4M urea.

Immunoglobulin A1 protease (iga) nucleotide sequence [Streptococcus pneumoniae TIGR4] (SEQ ID NO: 49) ATGGAAAAGTATTTTGGTGAAAAACAAGAGCGTTTTTCATTTAGAAAATT ATCAGTAGGACTTGTATCTGCAACGATTTCAAGTTTATTTTTTATGTCTG TATTAGCTAGTTCATCTGTGGATGCTCAAGAAACTGCGGGAGTTCACTAT AAATATGTGGCAGATTCAGAGCTATCATCAGAAGAAAAGAAGCAGCTTGT CTATGATATTCCGACATACGTGGAGAATGATGATGAAACTTATTATCTTG TTTATAAGTTAAATTCTCAAAATCAACTGGCGGAATTGCCAAATACTGGA AGCAAGAATGAGAGGCAAGCCCTAGTTGCTGGTGCTAGCTTAGCTGCTAT GGGAATTTTAATTTTTGCTGTTTCCAAGAAAAAGGTTAAGAATAAAACGG TATTACATTTAGTATTGGTTGCAGGGATAGGAAATGGTGTCTTAGTTTCA GTCCATGCTTTAGAAAATCATCTTTTGCTAAATTACAATACGGACTATGA ATTGACCTCTGGAGAAAAATTACCTCTTCCTAAAGAGATTTCAGGTTACA CTTATATTGGATATATCAAAGAGGGAAAAACGACTTCTGAGTCTGAAGTA AGTAATCAAAAGAGTTCAGTTGCCACTCCTACAAAACAACAAAAGGTGGA TTATAATGTTACACCGAATTTTGTAGACCATCCATCAACAGTACAAGCTA TTCAGGAACAAACACCTGTTTCTTCAACTAAGCCGACAGAAGTTCAAGTA GTTGAAAAACCTTTCTCTACTGAATTAATCAATCCAAGAAAAGAAGAGAA ACAATCTTCAGATTCTCAAGAACAATTAGCCGAACATAAGAATCTAGAAA CGAAGAAAGAGGAGAAGATTTCTCCAAAAGAAAAGACTGGGGTAAATACA TTAAATCCACAGGATGAAGTTTTATCAGGTCAATTGAACAAACCTGAACT CTTATATCGTGAGGAAACTATGGAGACAAAAATAGATTTTCAAGAAGAAA TTCAAGAAAATCCTGATTTAGCTGAAGGAACTGTAAGAGTAAAACAAGAA GGTAAATTAGGTAAGAAAGTTGAAATCGTCAGAATATTCTCTGTAAACAA GGAAGAAGTTTCGCGAGAAATTGTTTCAACTTCAACGACTGCGCCTAGTC CAAGAATAGTCGAAAAAGGTACTAAAAAAACTCAAGTTATAAAGGAACAA CCTGAGACTGGTGTAGAACATAAGGACGTACAGTCTGGAGCTATTGTTGA ACCCGCAATTCAGCCTGAGTTGCCCGAAGCTGTAGTAAGTGACAAAGGCG AACCAGAAGTTCAACCTACATTACCCGAAGCAGTTGTGACCGACAAAGGT GAGACTGAGGTTCAACCAGAGTCGCCAGATACTGTGGTAAGTGATAAAGG TGAACCAGAGCAGGTAGCACCGCTTCCAGAATATAAGGGTAATATTGAGC AAGTAAAACCTGAAACTCCGGTTGAGAAGACCAAAGAACAAGGTCCAGAA AAAACTGAAGAAGTTCCAGTAAAACCAACAGAAGAAACACCAGTAAATCC AAATGAAGGTACTACAGAAGGAACCTCAATTCAAGAAGCAGAAAATCCAG TTCAACCTGCAGAAGAATCAACAACGAATTCAGAGAAAGTATCACCAGAT ACATCTAGCAAAAATACTGGGGAAGTGTCCAGTAATCCTAGTGATTCGAC AACCTCAGTTGGAGAATCAAATAAACCAGAACATAATGACTCTAAAAATG AAAATTCAGAAAAAACTGTAGAAGAAGTTCCAGTAAATCCAAATGAAGGC ACAGTAGAAGGTACCTCAAATCAAGAAACAGAAAAACCAGTTCAACCTGC AGAAGAAACACAAACAAACTCTGGGAAAATAGCTAACGAAAATACTGGAG AAGTATCCAATAAACCTAGTGATTCAAAACCACCAGTTGAAGAATCAAAT CAACCAGAAAAAAACGGAACTGCAACAAAACCAGAAAATTCAGGTAATAC AACATCAGAGAATGGACAAACAGAACCAGAACCATCAAACGGAAATTCAA CTGAGGATGTTTCAACCGAATCAAACACATCCAATTCAAATGGAAACGAA GAAATTAAACAAGAAAATGAACTAGACCCTGATAAAAAGGTAGAAGAACC AGAGAAAACACTTGAATTAAGAAATGTTTCCGACCTAGAGTTATACAGTT TGTCAAATGGTACTTATAAACAACACATTTCGTTAGAGCAAGTTCCAAGC AATCCAAATAGCTACTTTGTTAAAGTGAAATCTTCTTCATTCAAAGATGT ATACCTACCAGTAGCATCAATATCAGAGGAAAGAAAAAATGATAAAATCC TTTATAAAATCACAGCAAAAGTAGAGAAGCTTCAGCAGGAGATAGAAAGC AGATATAAAGATAATTTTACCTTCTATCTAGCTAAGAAGGGAACAGAAGA AACAACAAACTTTACTTCCTTTAGTAATCTGGTCAAAGCTATAAACCAAA ATCCCTCTGGAACCTATCATTTAGCGGCCAGCCTGAATGCTAACGAAGTG GAGCTTGGTCCTGATGAAAGATCCTATATCAAGGACACCTTTACTGGTCG TTTAATCGGTGAAAAAGATGGCAAGAATTATGCTATCTATAATTTGAAAA AACCTCTGTTTGAAAACTTGAGTGGTGCTACAGTAGAAAAACTGAGTCTA AAAAATGTTGCTATTTCAGGGAAAGATGATATCGGTTCACTGGCAAATGA AGCTCAGAATAACACAAAAATTAAGCAAGTTCACGTCGATGGTGTTCTGG CTGGTGAACGTGGTATCGGTGGTTTGCTGGCTAAGGCTGAGCAATCAAGC ATCACAGAGAGCAGTTTCAAGGGAAGAATTATCAACACTTATGAAACGAC TGCTGCCTACAATATCGGTGGTATGGTCGGTCATTTGACAGGTGACAAGG CTTTACTTACTAAGTCAAAAGCGACAGTAGCCATTTCATCTAACACAAAT ACTTCAGATCAGACTGTGGGTGGACTTGCAGGCCTAGTAGACCGAGATGC ACAGATCCAAGATAGCTATGCTGAAGGTGATATCAACAATGTCAAGCACT TTGGTAGAGTCGCTGGAGTGGCAGGCAATTTGTGGGATCGAACTTCTGGT GATGTTAGGCATGCTGGAAGTTTGACCAATGTTCTCAGCGATGTTAATGT AACCAACGGAAATGCCATCACTGGTTACCACTATAACGAAATGAAGGTAA AGGACACATTCAGCAGCAAGGCCAACAGAGTCTACAATGTCACCTTGGTC AAGGATGAGGTCGTCAGCAAGGAATCCTTTGAAGAAAGAGGAACAATGCT AGATGCTTCTCAAATTGCAAGCAAAAAAGCAGAAATCAATCCTCTCATTT TACCAACAGTGGAGCCACTTTCAACAAGTGGCAAAAAAGACAGTGATTTT TCTAAGGTGGCCTATTATCAAGCTAAGCGCAACTTGACTTATAAAAACAT TGAAAAATTGCTACCTTTCTACAACAAGGCAACCATCGTCAAATACGGAA ACCTGGTCAATGAGAACAGTCTTTTATATCAAAAAGAACTCTTGTCAGCA GTCATGATGAAGGACAACCAAGTCATCACAGACATTGTTTCTAACAAACA GACTGCAAACAAACTCTTGCTTCACTACAAGGATGATTTATCTGAGAAGC TGGATCTCAAATACCAGAATGATTTCGCCAAATTAGCAGAATATAGTCTG GGCAATACTGGACTTCTCTATACGCCAAACCAATTCCTGTATGACCAAAC CTCTATCATCAAGCAAGTCTTACCTGACTTACAAAAGGTTGACTATCATT CAGAAGCCATCAGAAAGACGCTGGGTATTTCTCCAAACGTCAAGCAAACT GAGCTCTATCTAGAAGACCAGTTCGCCAAAACAAAACAACAACTGGAAGA CAGTTTGAAAAAACTCTTGTCAGCGGATGCTGGACTGGCTAGTGCTAACC CCGTCACTGAAGGTTATCTTGTAGATAAAATCAAACGCAACAAGGAAGCC TTGCTACTTGGCTTGACCTATCTGGAACGGTGGTATAACTTTAGCTATGG TCAGGTGAATGTCAAAGACCTAGTTCTGTACCATTTGGACTTCTTTGGTA AGGGGAATGCTTCACCATTAGATACTCTGATTGAGTTGGGTAAATCTGGC TTTAACAATCTTCTAGCTAAGAATAATGTCGATACTTATGGTATCAGTCT TGCCAGTCAACATGGAACGACAGATTTGTTTAGCACGCTGGAACATTACC GAAAAGTCTTTTTACCAAATACAAGCAATAATGACTGGTTTAAATCAGAG ACTAAGGCTTACATTGTCGAAGAAAAATCCACTATCGAAGAGGTGAAAAC GAAGCAAGGGTTAGCTGGCACCAAGTATTCTATCGGTGTTTATGATCGTA TCACGAGTGCCACATGGAAATACCGCAATATGGTCTTGCCTCTCCTGACC TTGCCAGAGAGATCCGTATTTGTCATCTCGACCATGTCTAGTCTAGGATT TGGAGCTTATGATCGCTACCGCAGTAGTGACCATAAAGCGGGCAAGGCTC TCAATGATTTTGTTGAAGAAAATGCGCGTGAAACAGCCAAACGTCAGCGA GATCACTACGATTATTGGTATCGTATTTTAGACGACAATGCACGTGAAAA ACTTTATAGAAATATTTTGCTTTACGATGCTTATAAATTTGGCGATGATA ATACCGTAGGGAAAGCTACAGAAGTGGCAGATTTTGATAATCCAAATCCT GCAATGCAACATTTCTTTGGACCTGTTGGAAATAAAGTTGGGCATAATCA ACACGGTGCTTATGCTACAGGTGATGCAGTTTATTATATGGGTTATCGAA TGTTGGATAAGGATGGAGCTATTACTTATACGCATGAGATGACACATGAC TCAGATCAGGACATTTATCTTGGAGGATATGGTCGAAGAAGTGGCTTGGG ACCAGAGTTCTTTGCTAAAGGATTATTACAAGCACCAGACCATCCAGATG ATGCGACCATTACCATCAACTCCATCTTGAAACATTCAAAATCTGATAGT ACAGAAAGTCGACGATTACAAGTACTTGATCCAACTACAAGATTTAATAA TGCAGATGATTTGAAGCAATATGTCCACAACATGTTTGACGTTGTTTATA TGTTGGAATATCTCGAAGGAAATTCAATTCTTAAATTGGATACGAATCAA AAACAACAACTTCTTAGAAAAGTTACAAATGAGTACCATCCTGATCCTGA TGGAAATAAGGTCTATGCAACAAATGTTGTCAGAAATCTAACAGTAGAAG AAGTTGAAAGACTACGTTCATTCAATGATTTGATTGATAATAATATTCTT TCGTCTAGGGAATATGCCTCAGGTAAATACGAAAGAAATGGCTACTTCAC TATTAAGTTATTTGCACCGATTTATGCTGCATTAAGTAATGATATAGGAA CACCAGGTGACCTGATGGGACGTCGTATAGCCTATGAACTACTAGCTGCT AAAGGCTTTAAAGATGGTATGGTACCATATATCTCAAACCAATACGAAGA AGAAGCCAAACAAAAGGGCAAGACAATCAATCTCTACGGTAAAACAAGAG GTTTGGTTACAGATGACTTGGTTTTGGAAAAGGTATTTAATAACCAATAT CATACTTGGAGTGAGTTTAAGAAAGCTATGTATCAAGAACGACAAGATCA GTTTGATAGATTGAACAAAGTTACTTTTAATGATACAACACAGCCTTGGC AAACATTTGCCAAGAAAACTACAAGCAGTGTAGATGAATTACAGAAATTA ATGGACGTTGCTGTTCGTAAGGATGCAGAACACAATTACTACCATTGGAA TAACTACAATCCAGACATAGATAGTGAAGTCCACAAGCTCAAGAGAGCAA TCTTTAAAGCCTATCTTGACCAAACAAATGATTTTAGAAGTTCAATTTTT GAGAATAAAAAATAG Amino Acid sequence Iga (SEQ ID NO: 51) MEKYFGEKQERFSFRKLSVGLVSATISSLFFMSVLASSSVDAQETAGVHY KYVADSELSSEEKKQLVYDIPTYVENDDETYYLVYKLNSQNQLAELPNTG SKNERQALVAGASLAAMGILIFAVSKKKVKNKTVLHLVLVAGIGNGVLVS VHALENHLLLNYNTDYELTSGEKLPLPKEISGYTYIGYIKEGKTTSESEV SNQKSSVATPTKQQKVDYNVTPNFVDHPSTVQAIQEQTPVSSTKPTEVQV VEKPFSTELINPRKEEKQSSDSQEQLAEHKNLETKKEEKISPKEKTGVNT LNPQDEVLSGQLNKPELLYREETMETKIDFQEEIQENPDLAEGTVRVKQE GKLGKKVEIVRIFSVNKEEVSREIVSTSTTAPSPRIVEKGTKKTQVIKEQ PETGVEHKDVQSGAIVEPAIQPELPEAVVSDKGEPEVQPTLPEAVVTDKG ETEVQPESPDTVVSDKGEPEQVAPLPEYKGNIEQVKPETPVEKTKEQGPE KTEEVPVKPTEETPVNPNEGTTEGTSIQEAENPVQPAEESTTNSEKVSPD TSSKNTGEVSSNPSDSTTSVGESNKPEHNDSKNENSEKTVEEVPVNPNEG TVEGTSNQETEKPVQPAEETQTNSGKIANENTGEVSNKPSDSKPPVEESN QPEKNGTATKPENSGNTTSENGQTEPEPSNGNSTEDVSTESNTSNSNGNE EIKQENELDPDKKVEEPEKTLELRNVSDLELYSLSNGTYKQHISLEQVPS NPNSYFVKVKSSSFKDVYLPVASISEERKNDKILYKITAKVEKLQQEIES RYKDNFTFYLAKKGTEETTNFTSFSNLVKAINQNPSGTYHLAASLNANEV ELGPDERSYIKDTFTGRLIGEKDGKNYAIYNLKKPLFENLSGATVEKLSL KNVAISGKDDIGSLANEAQNNTKIKQVHVDGVLAGERGIGGLLAKAEQSS ITESSFKGRIINTYETTAAYNIGGMVGHLTGDKALLTKSKATVAISSNTN TSDQTVGGLAGLVDRDAQIQDSYAEGDINNVKHFGRVAGVAGNLWDRTSG DVRHAGSLTNVLSDVNVTNGNAITGYHYNEMKVKDTFSSKANRVYNVTLV KDEVVSKESFEERGTMLDASQIASKKAEINPLILPTVEPLSTSGKKDSDF SKVAYYQAKRNLTYKNIEKLLPFYNKATIVKYGNLVNENSLLYQKELLSA VMMKDNQVITDIVSNKQTANKLLLHYKDDLSEKLDLKYQNDFAKLAEYSL GNTGLLYTPNQFLYDQTSIIKQVLPDLQKVDYHSEAIRKTLGISPNVKQT ELYLEDQFAKTKQQLEDSLKKLLSADAGLASANPVTEGYLVDKIKRNKEA LLLGLTYLERWYNFSYGQVNVKDLVLYHLDFFGKGNASPLDTLIELGKSG FNNLLAKNNVDTYGISLASQHGTTDLFSTLEHYRKVFLPNTSNNDWFKSE TKAYIVEEKSTIEEVKTKQGLAGTKYSIGVYDRITSATWKYRNMVLPLLT LPERSVFVISTMSSLGFGAYDRYRSSDHKAGKALNDFVEENARETAKRQR DHYDYWYRILDDNAREKLYRNILLYDAYKFGDDNTVGKATEVADFDNPNP AMQHFFGPVGNKVGHNQHGAYATGDAVYYMGYRMLDKDGAITYTHEMTHD SDQDIYLGGYGRRSGLGPEFFAKGLLQAPDHPDDATITINSILKHSKSDS TESRRLQVLDPTTRFNNADDLKQYVHNMFDVVYMLEYLEGNSILKLDTNQ KQQLLRKVTNEYHPDPDGNKVYATNVVRNLTVEEVERLRSFNDLIDNNIL SSREYASGKYERNGYFTIKLFAPIYAALSNDIGTPGDLMGRRIAYELLAA KGFKDGMVPYISNQYEEEAKQKGKTINLYGKTRGLVTDDLVLEKVFNNQY HTWSEFKKAMYQERQDQFDRLNKVTFNDTTQPWQTFAKKTTSSVDELQKL MDVAVRKDAEHNYYHWNNYNPDIDSEVHKLKRAIFKAYLDQTNDFRSSIF ENKK Recombinant His Tagged Iga MGQTEPEPSNGNSTEDVSTESNTSNSNGNEEIKQENELDPDKKVEEPEKT LELRNVSDLELYSLSNGTYKQHISLEQVPSNPNSYFVKVKSSSFKDVYLP VASISEERKNDKILYKITAKVEKLQQEIESRYKDNFTFYLAKKGTEETTN FTSFSNLVKAINQNPSGTYHLAASLNANEVELGPDERSYIKDTFTGRLIG EKDGKNYAIYNLKKPLFENLSGATVEKLSLKNVAISGKDDIGSLANEAQN NTKIKQVHVDGVLAGERGIGGLLAKAEQSSITESSFKGRIINTYETTAAY NIGGMVGHLTGDKALLTKSKATVAISSNTNTSDQTVGGLAGLVDRDAQIQ DSYAEGDINNVKHFGRVAGVAGNLWDRTSGDVRHAGSLTNVLSDVNVTNG NAITGYHYNEMKVKDTFSSKANRVYNTLVKDEVVSKESFEERGTMLDASQ IASKKAEINPLILPTVEPLSTSGKKDSDFSKVAYYQAKRNLTYKNIEKLL PFYNKATIVKYGNLVNENSLLYQKELLSAVMMKDNQVITDIVSNKQTANK LLLHYKDDLSEKLDLKYQNDFAKLAEYSLGNTGLLYTPNQFLYDQTSIIK QVLPDLQKVDYHSEAIRKTLGISPNVKQTELYLEDQFAKTKQQLEDSLKK LLSADAGLASANPVTEGYLVDKIKRNKEALLLGLTYLERWYNFSYGQVNV KDLVLYHLDFFGKGNASPLDTLIELGKSGFNNLLAKNNVDTYGISLASQH GTTDLFSTLEHYRKVFLPNTSNNDWFKSETKAYIVEEKSTIEEVKTKQGL AGTKYSIGVYDRITSATWKYRNMVLPLLTLPERSVFVISTMSSLGFGAYD RYRSSDHKAGKALNDFVEENARETAKRQRDHYDYWYRILDDNAREKLYRN ILLYDAYKFGDDNTVGKATEVADFDNPNPAMQHFFGPVGNKVGHNQHGAY ATGDAVYYMGYRMLDKDGAITYTHEMTHDSDQDIYLGGYGRRSGLGPEFF AKGLLQAPDHPDDATITINSILKHSKSDSTESRRLQVLDPTTRFNNADDL KQYVHNMFDVVYMLEYLEGNSILKLDTNQKQQLLRKVTNEYHPDPDGNKV YATNVVRNLTVEEVERLRSFNDLIDNNILSSREYASGKYERNGYFTIKLF APIYAALSNDIGTPGDLMGRRIAYELLAAKGFKDGMVPYISNQYEEEAKQ KGKTINLYGKTRGLVTDDLVLEKVFNNQYHTWSEFKKAMYQERQDQFDRL NKVTFNDTTQPWQTFAKKTTSSVDELQKLMDVAVRKDAEHNYYHWNNYNP DIDSEVHKLKRAIFKAHHHHHH PsaA

The gene for PsaA was amplified by PCR from Strepococcus pneumoniae D39 with the primers 5′CCATGGCTAGCCACCATCACCATCACCATGCTACAAACTCAATCATCGC 3′ (SEQ ID NO: 52) and 5′TTTCGGATCCTTATTTGCCAATCCTTCAGC 3′ (SEQ ID NO: 53) which incorporate flanking NheI and BamHI restriction sites, and a N-terminal His6 tag. The amplified DNA was cloned into a pET11a expression vector (Stratagene, LaJolla, Calif.) and electrotransformed into E. coli BL21(DE3). The recombinant protein was purified by Ni ⁺ affinity chromatography with the HisTrap Kit (Amersham Pharmacia) according to the manufacturers recommendations. To prevent the formation of inclusion bodies, 8 M urea was added to all buffers throughout the purification process. The purified recombinant protein was dialysed against 10 mM HEPES buffer, pH7.5, free dried, and stored at −20° C.

psaA nucleotide sequence Streptococcus pneumoniae D39 (SEQ ID NO: 54) ATGAAAAAATTAGGTACATTACTCGTTCTCTTTCTTTCTGCAATCATTCT TGTAGCATGTGCTAGCGGAAAAAAAGATACAACTTCTGGTCAAAAACTAA AAGTTGTTGCTACAAACTCAATCATCGCTGATATTACTAAAAATATTGCT GGTGACAAAATTGACCTTCATAGTATCGTTCCGATTGGGCAAGACCCACA CGAATACGAACCACTTCCTGAAGACGTTAAGAAAACTTCTGAGGCTGATT TGATTTTCTATAACGGTATCAACCTTGAAACAGGTGGCAATGCTTGGTTT ACAAAATTGGTAGAAAATGCCAAGAAAACTGAAAACAAAGACTACTTCGC AGTCAGCGACGGCGTTGATGTTATCTACCTTGAAGGTCAAAATGAAAAAG GAAAAGAAGACCCACACGCTTGGCTTAACCTTGAAAACGGTATTATTTTT GCTAAAAATATCGCCAAACAATTGAGCGCCAAAGACCCTAACAATAAAGA ATTCTATGAAAAAAATCTCAAAGAATATACTGATAAGTTAGACAAACTTG ATAAAGAAAGTAAGGATAAATTTAATAAGATCCCTGCTGAAAAGAAACTC ATTGTAACCAGCGAAGGAGCATTCAAATACTTCTCTAAAGCCTATGGTGT TCCAAGTGCCTACATCTGGGAAATCAATACTGAAGAAGAAGGAACTCCTG AACAAATCAAGACCTTGGTTGAAAAACTTCGCCAAACAAAAGTTCCATCA CTCTTTGTAGAATCAAGTGTGGATGACCGTCCAATGAAAACTGTTTCTCA AGACACAAACATCCCAATCTACGCACAAATCTTTACTGACTCTATCGCAG AACAAGGTAAAGAAGGCGACAGCTACTACAGCATGATGAAATACAACCTT GACAAGATTGCTGAAGGATTGGCAAAATAA PsaA Amino acid sequence (SEQ ID NO: 55) MKKLGTLLVLFLSAIILVACASGKKDTTSGQKLKVVATNSIIADITKNIA GDKIDLHSIVPIGQDPHEYEPLPEDVKKTSEADLIFYNGINLETGGNAWF TKLVENAKKTENKDYFAVSDGVDVIYLEGQNEKGKEDPHAWLNLENGIIF AKNIAKQLSAKDPNNKEFYEKNLKEYTDKLDKLDKESKDKFNKIPAEKKL IVTSEGAFKYFSKAYGVPSAYIWEINTEEEGTPEQIKTLVEKLRQTKVPS LFVESSVDDRPMKTVSQDTNIPIYAQIFTDSIAEQGKEGDSYYSMMKYNL DKIAEGLAK Recombinant His tagged PsaA (SEQ ID NO: 56) MAHHHHHHTNSIIADITKNIAGDKIDLHSIVPIGQDPHEYEPLPEDVKKT SEADLIFYNGINLETGGNAWFTKLVENAKKTENKDYFAVSDGVDVIYLEG QNEKGKEDPHAWLNLENGIIFAKNIAKQLSAKDPNNKEFYEKNLKEYTDK LDKLDKESKDKFNKIPAEKKLIVTSEGAFKYFSKAYGVPSAYIWEINTEE EGTPEQIKTLVEKLRQTKVPSLFVESSVDDRPMKTVSQDTNIPIYAQIFT DSIAEQGKEGDSYYSMMKYNLDKIAEGLAK

ELISA test for antibodies

IgG antibodies to Eno, Iga, PpmA and SlrA in serum of humans suffering from pneumococcus infection were measured by EIA. Microtiter plates (Maxisorp; Nunc, Roskilde, Denmark) were coated with 100 μl/well, with a coating concentration of 5 μg/ml of antigen in PBS and incubated overnight at 37° C. The plates were blocked with 1% bovine serum albumin (BSA) and 0.05% Tween 20 in PBS at 37° C. for 1 h. The samples were serially diluted (starting at 1:100) in the dilution buffer (1% BSA, 0.05% Tween 20 in PBS). Duplicates (100 μl/well) were incubated at 370° C. for 2 h. Polyclonal alkaline phosphatase-conjugated antisera to human IgG (Sigma Chemicals, St. Louis) was diluted 1:3000 in the dilution buffer, pipetted at 100 μl/well, and incubated for 2 h at 37° C. Finally, 100 μl/well of MUP (methylumbelliferyl phosphate) (Sigma, CAT#M8883) substrate solution (0.2 mM 4-methylumbelliferyl phosphate, 0.05 M NaCO3, 0.05 mM MgC12) was added, and the plates were incubated for 1 h at 37° C. The fluorescence was measured with an excitation wavelength of 365 nm, and an emission wavelength of 450 nm with a Molecular Devices SpectraMAX (Sunnyvale, USA) microplate reader. The serum endpoint antibody titer was interpolated from the intersection of the absorbance-versus-serum dilution curve at the background fluorescence plus four times the standard deviation. The end-point titer was then converted into antibody concentration (EIA units of immunoglobulin per milliliter) by comparison with a standard serum consisting of pooled healthy adult volunteers, and was considered to contain 100 U/mL of anti-a-enolase, Iga, PpmA or SlrA. Samples with antibody concentrations below the detection limit (30 U/mL for Eno, 4 U/mL for Iga, 4 U/mL for SlrA, and 5 U/mL for PpmA) were assigned the values equivalent to half the detection limit. The results are shown in Table 9.

Statistical methods: We used SPSS software (SPSS, USA) for analysis of log-transformed data. The antibody concentrations in the groups of children are reported as geometric mean concentrations . (GMC, units/ml) with 95% confidence intervals (95% CI) and compared by one way ANOVA (analysis of variable) followed by a LSD (least significant difference) post hoc test. Statistical comparisons between antibody levels in acute and convalescent phase sera were carried out using a paired Student's t test. Fisher's exact test was used when the proportions of variables were compared. A logistic regression was used to evaluate the effects of the acute phase antibody concentration, age and the nature of pneumococcal contact on the antibody response and to evaluate antibody concentrations (logarithm base e of antibody concentration) as a risk factor for the involvement of pneumococci as a risk factor for the development of AOM (acute otitis media). Children were grouped into eight 3-month groups according to the age at which the acute serum was sampled. To determine whether there were any significant differences in relation to age, groups were analyzed by 1-way ANOVA followed by a Tukey's HSD (honestly significance difference) test.

TABLE 9 Results of ELISA test t = 0 t = final (pre-immune (post-immune serum) serum) average SD average SD PpmA PpmA 0.11 0.41 5147.51 4576.27 PpmA + Ply 0 0 2796.24 1615.74 PpmA + PsaA 0.08 0.31 3700.60 2553.09 PpmA + Enolase 0.39 0.59 2388.10 1397.90 PpmA + SlrA 0.42 0.59 3407.62 2696.83 PpmA + TetTox. 0.57 0.52 881.93 813.19 PpmA + IgA1 0.38 1.22 6902.50 5195.41 PpmA + PsaA + IgA1 0.90 1.24 2008.46 1285.48 SlrA SlrA 0 0 3897.68 4180.47 SlrA + PpmA 0 0 2026.06 1083.99 IgA1 IgA1 0.13 0.80 16365.08 18891.05 IgA1 + PpmA 0.17 0.55 16291.67 10028.60 IgA1 + PsaA 0 0 19452.50 13428.11 IgA1 + PpmA + PsaA 0 0 14177.78 7133.86 PsaA PsaA 2.63 4.47 20456.25 21898.98 PsaA + PpmA 0.81 1.82 51793.75 45759.81 PsaA + IgA1 0 0 42600.00 52685.55 PsaA + PpmA + IgA1 1.71 2.22 25805.56 25713.50

EXAMPLE 2

Mucosal Vaccinations

Lactococcus lactis ghosts in combination with the AcmA cell-wall-binding domain (protein anchor) can be used as a carrier/adjuvant system for mucosal and systemic delivery of antigens (WO 99/25836 and WO 02/101026).

In the present study immunogenicity and protective activity of a tri-valent ghost-protein vaccine (SlrA, Iga1prt, PpmA) was tested as well as that of each of the monovalent ghost-protein vaccines after nasal administration of the vaccines and nasal challenge with virulent S. pneumoniae. The aim of the experiments was:

-   -   To evaluate protective activity of an intranasal administered L.         lactis ghost-protein anchor tri-valent (SlrA, PpmA, Iga1prt)         vaccine and of ghost-protein anchor mono-valent (SlrA),         ghost-protein anchor mono-valent (PpmA) and ghost-protein anchor         mono-valent (Iga1prt) vaccines.     -   To evaluate the immune response to intranasal administered         antigens bound to ghost lactococci and compare the immune         response to the antigens administered simultaneously or         individually.     -   To evaluate correlation of bacterial load in lungs, blood and         nasopharynx with protection and immune response after         vaccination.         Materials and Methods

Construction of vectors. Table 10 summarises the characteristics of the antigens used in the ghost protein-based S. pneumoniae vaccines.

PpmA::PA fusion. Plasmid pPA32, specifying a fusion between the S. pneumoniae PpmA protein and the protein anchor (FIG. 12), was constructed by ligating an NcoI- and EcoRI-cleaved PCR-amplified fragment of CbpA into the NcoI and EcoRI sites of pPA3 (Steen et al. 2003. J. Biol. Chem. 278:23874-23881). The PpmA-specific fragment was amplified with the primers PpmA.1 (cggtctcacatgtcgaaagggtcagaaggtgcagacc) and PpmA.2 (cggtctcgaattgcttcgtttgatgtactactgcttgag) with S. pneumoniae strain D39 chromosomal DNA as template. The ligation mix was cut with EcoRI and NheI before it was used for electroporation of L. lactis PA1001 (Kuipers et al. J. Biol. Chem. 2004 Mar. 24 [Epub ahead of print]). The final construct was designated pPA32.

Iga1prt::PA fusion. Plasmid pPA152, specifying a fusion between a fragment of the S. pneumoniae Iga1prt protein (residues 672 to 1330) and the protein anchor (FIG. 13). Plasmid pPA152 was constructed by ligating an NcoI- and EcoRI-cleaved PCR-amplified fragment of Iga1prt into the corresponding sites of pPA3. The Iga1prt-specific fragment was amplified with the primers Iga1.fw2 (cgtctcccatgggacaaacagaaccagagccatcaaacgg) and Iga1trunc2.rev (ccgtctcgaattctagccagtccagcatc) with S. pneumoniae strain TIGR4 chromosomal DNA as template. The ligation mix was cut with NheI before it was used for electroporation of L. lactis PA1001. The final construct was designated pPA152.

SlrA::PA fusion. Plasmid pPA162, specifying a fusion between the S. pneumoniae SlrA protein and the protein anchor (FIG. 14), was constructed by ligating an Esp3I-cleaved PCR-amplified fragment of SlrA into the NcoI and EcoRI sites of plasmid pPA3. The SlrA-specific fragment was amplified with the primers SlrA.fw (ccgtctcccatggtccaacgcagtctgcgt) and SlrA.rev (ccgtctcgaattccagatttaaaatcgtagtctttcacca) with S. pneumoniae strain D39 chromosomal DNA as template. The ligation mix was cut with SphI before it was used for electroporation of L. lactis PA1001. The final construct was designated pPA162.

Production of Ghost cells. L. lactis Ghosts were produced as described in WO 02/101026. Each dosis representing the amount used for one nasal administration in mice, contained approximately 2.5×109 Ghosts.

Production of antigen. The yeast-based GLS medium was used for production of the vaccine. GLS contains 2% gistex (Gistex® LS Ferm Powder AGGL -1027180, Strik Special Additives, The Netherlands), 3% β-glycerophosphate and 2% glucose. Glucose was sterilized as a separate solution and added to the broth when the strain was inoculated. L. lactis PA1001 containing plasmids pPA152, pPA32 or pPA162 were grown in GLS supplemented with 5 μg/ml chloramphenicol. At OD600 levels of approximately 0.5 and 1.0 the cultures were induced by adding 1 ml of the cell-free supernatant of a nisin producing L. lactis culture per liter of medium (1:1000; Kuipers et al. 1997. Tibtech. 15: 135-140). The culture was left at 30° C. overnight, after which the culture supernatants were concentrated with a VivaFlow tangential flow module (Vivascience VivaFlow 200; 10,000 Da cut-off). The concentrate was sterilised through a 0.45 μm filter and stored at +4° C. until binding.

Production of the vaccines. The vaccines were produced as follows:

Ghost-SlrA::PA monovalent vaccine:

-   27.5 ml of a 10× concentrated supernatant of a SlrA::PA producing L.     lactis culture was bound to 4.6 ml Ghost (46 units) on a blood     suspension mixer for 30 minutes at room temperature. After washing,     the pellet was resuspended in 920 μPBS and 880 μl was divided in 4     vials (220 μl each). 3 vials for the monovalent vaccine and 1 vial     for the trivalent vaccine.     Ghost-PpmA::PA Monovalent Vaccine: -   170 ml of a 10× concentrated supernatant of a PpmA::PA producing L.     lactis culture was bound to 4.6 ml Ghost (46 units) on a blood     suspension mixer for 30 minutes at room temperature. After washing,     the pellet was resuspended in 920 μl PBS and 880 μl was divided in 4     vials (220 μl each). 3 vials for the monovalent vaccine and 1 vial     for the trivalent vaccine.     Ghost-Iga1prt::PA Monovalent Vaccine: -   All concentrated supernatant (25-41×) of a Iga1prt::PA producing L.     lactis culture was bound to 4.6 ml Ghost (46 units) on a blood     suspension mixer for 30 minutes at room temperature. After washing,     the pellet was resuspended in 920 μl PBS and 880 μl was divided in 4     vials (220 μl each). 3 vials for the monovalent vaccine and 1 vial     for the trivalent vaccine.     Trivalent Vaccine: -   Three vials of monovalent vaccines were mixed and divided over 3     vials (220 μl each).

All vaccines were kept at −80° C. until the start of the immunizations. One dosis (20 μl) of a nasal vaccine contains 2.5×10⁹ Ghost cells.

Mice. Female outbred CD-1 mice were purchased from Harlan France. Mice were 6 week-old at the time of starting the study.

Pneumococcal challenge strain. S. pneumoniae D39, serotype 2, was obtained from the National Collection of Type Cultures (NCTC 7466; Central Public Health Laboratory, London).

Immunization and treatment groups. Mice were immunized intranasally under light inhalation anaesthesia (isoflurane/O₂/N₂O) by pipeting 20 μl of the vaccine or control onto the nostrils. Complete immunization consisted of 3 doses given at 10 day-intervals.

Mice (10 per group) received PBS as control. The vaccines consisted of 2.5×10⁹ ghosts and contained 5 μg Iga1prt, 10 □g PpmA and 50 μg SlrA per dose for the mono-valent vaccines, 2.5×10⁹ ghosts with a third of the monovalent doses of each antigen for the tri-valent vaccine.

The following groups were used:

-   -   1. 20 μl PBS.     -   2. 2.5×10⁹ lactococcal ghosts-protein anchor-SlrA fusion (50 μg         SlrA::PA) in 20 μl PBS.     -   3. 2.5×10⁹ lactococcal ghosts-protein anchor-PpmA fusion (10 μg         PpmA::PA) in 20 μl PBS.     -   4. 2.5×10⁹ lactococcal ghosts-protein anchor-IgAprt fusion         (IgAprt::PA) in 20 μl PBS.     -   5. 2.5×10⁹ lactococcal ghosts with bound protein         anchor-SlrA/PpmA/Iga1prt fusions (antigens individually bound as         in groups 2, 3 and 4 and mixed in ratio 1:1:1) in 20 μl PBS.

Challenge. Intranasal challenge was performed 4 weeks after the last immunization. Aliquots of inoculum were rapidly thawed and bacteria diluted in sterile PBS to 1×10⁶ CFU/50 μl. Challenge was carried out under light anesthesia by pipeting 50 μl of the inoculum onto the nostrils of the mice. After inoculation were laid on their backs until recovery. Mice were weighted before challenge and at t=44-h. Condition of the mice was monitored frequently (at least three times a day) and scored for the following criteria: hunched, starry coat, lethargic, moribund. After 44-h mice blood was collected under anesthesia and mice terminated by cervical dislocation. Nose washes and lungs were collected for quantitative microbiological culture.

Scoring of health status. Each animal was scored 44 h after infection according to the criteria given in Table 11. The maximum score per group of 10 animals is 40 (all animals dead). All animals healthy results in a score of 0. The disease index is the percentage of the maximum score.

Determination of bacterial load. Blood samples, lungs homogenates and nose washes were serially diluted and plated on agar with 5% sheep blood. Plates were incubated overnight at 37° C. before counting the colonies. Statistical analyses were performed using GraphPad Instat. Difference between groups were assessed by unpaired student t-test with log values of bacterial. Loss of weight was assessed by paired t-test. Comparison of disease index was performed by Mann-Whitney analyse. A P-value <0.05 is considered significant.

ELISA. Antigens (SlrA, PpmA and Iga1protease), purified by making use of his-tag purification, were coated in 96 well plates (2 μg/ml) in coating buffer (50mM sodium carbonate, pH9.6). Only Igalprotease was coated at 10 μg/ml in coating buffer. For the calibration curve rat anti-mouse kappa chain (BD Biosciences 559749, 0.5 mg/ml) was coated at 1/500 dilution in coating buffer. After incubation overnight at 4° C., the plates were rinsed 3 times with wash buffer (0.02% Tween 20 in PBS). The plates were blocked by adding 100 μl blocking buffer (1% BSA, 0.02% Tween 20 in PBS) in each well. The plates were incubated for 1 hour at room temperature. All blood samples were diluted 1/100 with blocking buffer, and serial dilutions (⅓) of these samples were performed in 100 μl blocking buffer. For the calibration curve a serial dilution of mouse IgG1 kappa light chain (Sigma M7894) was made, starting with 1 μg/ml. All samples were tested in duplo. After incubation for 2 hours at room temperature, the plates were rinsed 3 times with wash buffer. Antigen specific antibodies were detected by using goat anti-mouse IgG (Fc)-alkaline phosphatase conjugate (Sigma A7434) diluted 1/5000 in blocking buffer. The plates were rinsed two times with washing buffer and two times with double distilled H2O and were developed with PNPP (Sigma N2765, 1 mg/ml in 50 mM sodium carbonate, 1 mM MgCl2, pH9.6). After 30 minutes of incubation with pNPP, the reaction was stopped by addition of 50 μl NaOH (2M). The absorbance at 405 nm was recorded and the IgG concentration in the blood samples was determined by comparison with the calibration curve.

Results

Composition of the vaccine. The amounts of antigen-PA fusions in the different vaccines were determined using CBB-stained SDS-PAGE gels (example in FIG. 15), containing samples from different Ghost-antigen groups and a BSA protein standard. The established amounts were for the monovalent vaccines:

Ghost-SlrA::PA: 50 μg/dose Ghost-PpmA::PA 10 μg/dose Ghost-Iga1prt::PA  5 μg/dose

The trivalent vaccine was composed by mixing the monovalent vaccines in a ratio of 1:1:1 with the total number of ghost the same as in the monovalent vaccines (2.5×109) resulting in the following amounts of antigen:

Ghost-SlrA::PA:  17 μg/dose Ghost-PpmA::PA 3.3 μg/dose Ghost-Iga1prt::PA 1.7 μg/dose

The vaccines were formulated in PBS without additional adjuvant.

Vaccine Efficacy.

Health status. Intranasal vaccination with the tri-valent ghost vaccine resulted in protection against a nasal challenge with virulent S. pneumoniae. The vaccinated mice remained healthy as reflected by a disease index significantly lower when compared to the mice that received PBS (FIG. 16). No significant loss of weight was seen in this group (FIG. 17), indicating a significant improvement in health. Of the monovalent vaccines, SlrA and PpmA did show minor protective effects (FIG. 16). The PpmA monovalent vaccine group also showing no significant loss of weight (FIG. 17), indicating a better overall health then in the PBS control group. However, overall the animals in the trivalent vaccine group clearly showed the best health status after challenge.

Bacterial counts (see FIG. 18 and Table 12). In mice vaccinated with the tri-valent vaccine the number of bacteria in the lungs and nose was significantly reduced compared to tlie PBS control group (P-values<0.05). This was not the case when the mice were immunized with each one of the monovalent vaccines. Also, comparison between the tri-valent vaccine group and the monovalent vaccine groups shows significant differences in the bacterial counts in nose, lung and blood. Again, clearly the tri-valent vaccine group performed best,

Immune response. Serum IgG response were determined by ELISA after 3 immunizations in pre-challenge blood. FIG. 19 shows the serum IgG antigen specific responses generated in the different vaccine groups. The responses against SlrA, PpmA and Iga1prt in the monovalent vaccines are similar to the responses to these antigens in the tri-valent vaccines, indicating that the amounts of antigen in the tri-valent vaccine are sufficient to elicit serum antibody responses of this magnitude for these antigens. The combined response to the three antigens in the tri-valent vaccine is needed to obtain the protective effect, since each of the individual responses in the monovalent vaccine groups did not provide sufficient protection.

CONCLUSIONS

-   -   Nasal (mucosal) vaccination with the tri-valent SlrA, PpmA,         Iga1prt vaccine provided increased protection of the mice after         challenge with S. pneumoniae.     -   Nasal vaccination with the tri-valent vaccine resulted in         significant reduction in the number of bacteria in lungs and         nose.     -   Nasal immunization with the mono- and trivalent vaccines         resulted in significant antigen specific IgG levels in the blood         serum.     -   Nasal vaccination with the mono-valent vaccines had no         protective effect upon challenge with S. pneumoniae.

TABLE 10 Overview of S. pneumoniae antigen characteristics. The segments that were used for the actual protein anchor fusions are indicated. Size of Present in original PA fusion Protein protein Signal aa & Antigen Genbank/SwissProt function (aa) sequence percentage SlrA NP_345269 Peptidyl-prolyl 267 1-17 (lipo) 21-267 (92%) isomerase (chaperone) PpmA NP_345462 Peptidyl-prolyl 313 1-22 (lipo) 23-311 (92%) isomerase (chaperone) Iga1prt NP_345623 Iga1 protease 2004 1-36 or 42 672-1330 (33%) 

TABLE 11 Scoring overview of health status. Score Interpretation Parameters 0 Healthy / 1 A bit ill Starry coat and/of hunched + 2 III Starry coat and/of hunched ++ Lethargic + 3 Very ill Starry coat and/of hunched +++ Lethargic ++ 4 Dead /

TABLE 12 Statistical comparisons between various vaccines. P values < 0.05 are considered significant (indicated in bold). Lungs Nose Blood Ghost-SlrA::PA with 0.7185 0.2022 0.9156 PBS Ghost-PpmA::PA with 0.7096 0.9758 0.8406 PBS Ghost-Iga1::PA with 0.3527 0.7923 0.26 PBS Tri-valent Ghosts with 0.0498 <0.0001 0.167 PBS Tri-valent Ghosts with 0.0378 0.0465 0.2005 Ghost-SlrA::PA Tri-valent Ghosts with 0.1853 0.0026 0.3289 Ghost-PpmA::PA Tri-valent Ghosts with 0.0142 0.0023 0.021 Ghost-Iga1::PA

REFERENCES

-   Obaro S K. The new pneumococcal vaccine. Clin Microbiol Infect 2002;     8(10): 623-33. -   Darkes M J, Plosker G L. Pneumococcal conjugate vaccine (Prevnar;     PNCRM7): a review of its use in the prevention of Strepococcus     pneumoniae infection. Paediatr Drugs 2002; 4(9):609-30. -   Wuorimaa T, Kayhty H. Current state of pneumococcal vaccines. Scand     J Immunol 2002; 56(2):111-29. -   Lee L H, Lee C J, Frasch C E. Development and evaluation of     pneumococcal conjugate vaccines: clinical trials and control tests.     Crit Rev Microbiol 2002; 28(1):27-41. -   KIugman K P. Efficacy of pneumococcal conjugate vaccines and their     effect on carriage and antimicrobial resistance. Lancet Infect Dis     2001; 1(2):85-91. -   Rapola S, Jantti V, Haikala R, Syrjanen R, Carlone G M, Sampson J S,     Briles DE, Paton J C, Takala A K, Kilpi T M, Kayhty H. Natural     development of antibodies to pneumococcal surface protein A,     pneumococcal surface adhesin A, and pneumolysin in relation to     pneumococcal carriage and acute otitis media. J Infect Dis 2000;     182(4):1146-52. -   Rapola S, Kilpi T, Lahdenkari M, Makela P H, Kayhty H. Antibody     response to the pneumococcal proteins pneumococcal surface adhesin A     and pneumolysin in children with acute otitis media. Pediatr Infect     Dis J 2001; 20(5):482-7. -   Zysk G, Bethe G, Nau R, Koch D, Grafin Von Bassewitz V C, Heinz H P,     Reinert R R. Immune response to capsular polysaccharide and surface     proteins of Strepococcus pneumoniae in patients with invasive     pneumococcal disease.J Infect Dis 2003 Jan 15;187(2):330-3. -   McCool T L, Cate T R, Moy G, Weiser J N. The immune response to     pneumococcal proteins during experimental human carriage. J Exp Med     2002; 195(3):359-65. -   Overweg K, Pericone C D, Verhoef G G, Weiser J N, Meiring H D, De     Jong A P, De Groot R, Hermans P W. Differential protein expression     in phenotypic variants of Strepococcus pneumoniae. Infect Immun     2000; 68(8):4604-10. -   Overweg K, Kerr A, Sluijter M, Jackson M H, Mitchell T J, de Jong A     P, de Groot R, Hermans P W. The putative proteinase maturation     protein A of Strepococcus pneumoniae is a conserved surface protein     with potential to elicit protective immune responses. Infect Immun     2000; 68(7):4180-8.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Titres of anti-pneumococcal IgG against PpmA in children and adults.

FIG. 2. Titres of anti-pneumococcal IgG against pneumolysin in children and adults.

FIG. 3. Titres of anti-pneumococcal IgG against IgA1 proteinase in children and adults.

FIG. 4. Titres of anti-pneumococcal IgG against alpha-enolase in children and adults.

FIG. 5. Titres of anti-pneumococcal IgG against SirA in children and adults.

FIG. 6. Survival time of SirA-vaccinated CD-1 mice infected intranasally with Strepococcus pneumoniae D39.

FIG. 7. Survival time of IgA1 proteinase-vaccinated CD-1 mice infected intranasally with Strepococcus pneumnoniae D39.

FIG. 8. Survival time of PsaA-vaccinated CD-1 mice infected intranasally with Strepococcus pneumoniae D39.

FIG. 9. Survival time of SirA- and PpmA-vaccinated CD-1 mice infected intranasally with Strepococcus pneumoniae D39.

FIG. 10. Passive immunization of CD-1 mice with pre-immune serum (PIS), anti-PpmA serum (APS) and challenged intranasally with Strepococcus pneumoniae D39 or a PpmA-negative knockout strain of Strepococcus pneumoniae.

FIG. 11. Survival of MF-1 mice passively vaccinated with anti-PpmA antiserum and intranasally infected with Strepococcus pneumoniae D39.

FIG. 12. Sequence of the secreted PpmA::PA fusion protein. Underlined are the aminoacids of Iga1prt. Protein anchor (PA) amino acids are in italics.

FIG. 13. Sequence of the secreted Iga1prt::PA fusion protein. Underlined are the aminoacids of Iga1prt. Protein anchor (PA) amino acids are in italics.

FIG. 14. Sequence of the secreted SlrA::PA fusion protein. Underlined are the aminoacids of Iga1prt. Protein anchor (PA) amino acids are in italics.

FIG. 15. A CBB (Coomassie Brilliant Blue) stained SDS-PAGE gel with the Ghost-antigen vaccines and a BSA (bovine serum albumin) reference. SlrA, PpmA and Iga1 are samples from the vaccine groups. Each sample represents one dose (20 μl). In the five lanes on the right, the number represents the amount of BSA in micrograms. An estimate of the amount of bound antigen is made and shown in the lower right part of the Figure.

FIG. 16. Health status of the mice at the time of ending experiment (t=44-h after infection). A score ranging from 0-4 was attributed to each mouse according to the parameters described in material and methods. The disease index was calculated as percentage of maximal score and presented as average+SEM for each group. A high score indicates that most animals are diseased. A low score indicates that most animals are healthy.

FIG. 17. Weight loss upon challenge. Mice were weighted prior infection and 44-h after challenge. Loss of weight is calculated as percentage of the initial weight and presented as average+SEM for each group. Loss of weight is an indication that animals are diseased.

FIG. 18. Bacterial counts 44-h post-challenge in the lungs (A), nose (B) and blood (C). Graphs show individual data (⋄) and the average (—) for each group. High bacterial counts are an indication that animals are not protected.

FIG. 19. Antigen specific IgG in the serum after 3 immunizations but prior to challenge. The values are presented as average+SEM for each group. 

1. A composition for inducing an immune response to infection by Streptococcus pneumoniae, comprising at least an antigenic part of pneumococcal maturation protein A (PpmA), at least an antigenic part of streptococcal lipoprotein rotamase A (SlrA), and SEQ ID NO:
 62. 2. The composition according to claim 1, further comprising a pharmaceutically acceptable carrier and /or adjuvant.
 3. The composition according to claim 2, wherein said adjuvant is obtained from bacterial cell wall material.
 4. The composition according to claim 2, wherein said adjuvant is a ghost.
 5. The composition according to claim 4, wherein said ghost is a ghost from Lactococcus lactis.
 6. The composition according to claim 1, wherein said protein is coupled to a ghost.
 7. The composition according to claim 6, wherein said ghost is a ghost from Lactococcus lactis.
 8. The composition according to claim 6, wherein said coupling is effected by a protein anchor.
 9. The composition according to claim 8, wherein said protein anchor is AcmA.
 10. A method for treating an infection by Streptococcus pneumoniae comprising administering to a mammal in need thereof a composition, said composition comprises at least an antigenic part of pneumococcal maturation protein A (PpmA), at least an antigenic part of streptococcal lipoprotein rotamase A (SlrA), and SEQ ID NO:
 62. 11. A method for inducing an immune response to infection by Streptococcus pneumoniae comprising administering to a mammal in need thereof a composition, said composition comprises at least an antigenic part of pneumococcal maturation protein A (PpmA), at least an antigenic part of streptococcal lipoprotein rotamase A (SlrA), and SEQ ID NO:
 62. 12. A method according to claim 11, wherein the administration is mucosal.
 13. A method according to claim 11, wherein the administration is nasal. 